for reference purposes · 2020. 11. 24. · bioo scienti˜c corporation · 7050 burleson road,...

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Bioo Scienti�c Corporation · 7050 Burleson Road, Austin, Texas 78744 · BiooScientific.comP: 1.888.208.2246 · F: 512.707.8122 · Bioo Research Products Group · [email protected]

Made in the USA

THE NGS EXPERTS™

FOR REFERENCE PURPOSESThis manual is for Reference Purposes Only. DO NOT use

this protocol to run your assays. Periodically, optimizations

and revisions are made to the kit and protocol, so it is

important to always use the protocol included with the kit.

NEXTflex™ qRNA-Seq™ Kit v2 - Set D(Illumina Compatible)

Catalog #5130-15 (48 reactions)

©Bioo Scientific Corp. • 2015 V15.01

�is product is for research use only.

�is manual is proprietary to Bioo Scienti�c Corp., and intended only for customer use in connection with the product(s) described herein and for no other purpose. �is document and its contents shall not be used or distributed for any other purpose without the prior written consent of Bioo Scienti�c. Periodic optimizations and revisions are made to kit components and manu-als. Follow the protocol included with the kit.

Bioo Scienti�c makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. �ere is no warranty of merchantability for this product, or of the �tness of the product for any purpose. Bioo Scienti�c shall not be liable for any damages, including special or consequential damages, or expense arising directly or indirectly from the use of this product.

Bioo Scienti�c, NEXT�ex, AIR, �e NGS Experts, qRNA, and NanoQ are trademarks or registered trademarks of Bioo Scienti�c. All other brands and names contained herein are the property of their respective owners.

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THE NGS EXPERTS™ 1

NEXTflex™ qRNA-Seq™ Kit v2 - Set D - 5130-15

GENERAL INFORMATION 2Product Overview 2Revision History 2Contents, Storage and Shelf Life 3Required Materials not Provided 4Warnings and Precautions 5

NEXTflex™ qRNA-SEQ™ RNA SAMPLE PREPARATION PROTOCOL 6NEXTflex™ qRNA-Seq™ RNA Sample Preparation Flow Chart 6

NEXTflex™ qChIP-SEQ™ DNA SAMPLE PREPARATION PROTOCOL 7NEXTflex™ qChIP-Seq™ DNA Sample Preparation Flow Chart 7Starting Material 8

OPTION 1 – NEXTflex™ qRNA-SEQ™ 9STEP A1: RNA Fragmentation 9STEP B1: First Strand Synthesis 10STEP C1: Second Strand Synthesis 11STEP D1: Bead Cleanup 12STEP E1: End Repair 13STEP F1: Bead Cleanup 14STEP G1: Adenylation 15STEP H1: Adapter Ligation 16STEP I1: Bead Cleanup 17STEP J1: PCR Amplification 19STEP K1: Bead Cleanup 20

OPTION 1 – NEXTflex™ qRNA-SEQ™ LIBRARY VALIDATION 22OPTION 2 – NEXTflex™ qChIP-SEQ™ 23

STEP A2: End Repair 23STEP B2: Bead Cleanup 24STEP C2: Adenylation 25STEP D2: Adapter Ligation 26STEP E2: Bead Cleanup 27STEP F2: PCR Amplification 29STEP G2: Bead Cleanup 30

OPTION 2 – NEXTflex™ qCHIP-SEQ™ LIBRARY VALIDATION 32APPENDIX A 33

Molecular Index Adapters (A1-A96) 33NEXTflex™ qRNA-Seq™ Primer Sequences 34Low Level Multiplexing 34

RELATED PRODUCTS 35NOTES 38

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GENERAL INFORMATION

Product Overview

The NEXTflex™ qRNA-Seq™ Kit v2 is designed to prepare RNA, ChIP DNA, and genomic DNA samples for sequencing using Illumina® sequencing platforms, while enabling high precision measurement of starting material concentration. The kit streamlines the library preparation procedure required for paired-end and multiplexed sequencing.

The NEXTflex™ qRNA-Seq™ Kit v2 efficiently generates libraries equivalent to conventional RNA-Seq libraries, but with the added feature of Molecular Indexing™, a DNA labeling tech-nology developed by Cellular Research, Inc. This kit contains a set of 96 distinct molecular labels on the sequencing adapters. Each label consists of an 8 nucleotide barcode tag. During the ligation reaction, each cDNA fragment independently and randomly ligates to a single label from this pool of 96 adapters to result in a total of 96 x 96 = 9,216 possible combinations across both ends. For every clone sequenced, paired-end reads reveal the chosen label on each end along with adjoining cDNA sequence. This allows for differentiation between re-sampling of the same molecule and sampling of a different molecule of identical sequence. Analysis using molecular indexing information provides an absolute, digital measurement of gene expression levels or ChIP enrichment, irrespective of common amplification distortions observed in tradi-tional library preparation. In addition to encoding DNA fragments at the molecular level, the kit also allows for the application of sample-specific barcodes during the library preparation PCR step. A more detailed description of the use of molecular indexing is available in our product application note.

Molecular indexing technology can be applied to DNA and RNA starting material alike. In order to make molecular indexed DNA libraries, follow Option 2.

For deconvolution of unique read fragments, Bioo Scientific now offers a dqRNASeq script.Using read pairs aligned to transcripts and FASTQ files, this script will generate a table listing fragments, start/stop sites in transcripts (USS), and molecular labels (STL). The script will also generate a table listing the total number of read pairs per transcript and the number of read pairs after STL, USS, and STL/USS correction. The script can be downloaded here or contact us at [email protected].

Revision History

Version Date Description

V15.01 January 2015 Initial Product Launch

http://www.bioongs.comhttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-rapid-directional-qrna-seq-kit/resources#/LiveTabsContent68055366mailto:BiooNGS%40BiooScientific.com?subject=NEXTflex%20qRNA-Seq%20Script%20


Contents, Storage and Shelf Life

The NEXTflex™ qRNA-Seq™ Kit v2 contains enough material to prepare 48 libraries for Illu-mina® compatible sequencing. The shelf life of all reagents is 12 months when stored proper-ly. All components can safely be stored at -20°C.

Kit Contents AmountBROWN CAP

NEXTflex™ RNA Fragmentation Buffer 240 µL

RED CAP

NEXTflex™ First Strand Synthesis Primer 48 µL

NEXTflex™ First Strand Synthesis Buffer Mix 192 µL

BLUE CAP

NEXTflex™ Second Strand Synthesis Mix 2 (600) µL

PURPLE CAP

NEXTflex™ End Repair Buffer Mix 336 µL

NEXTflex™ End Repair Enzyme Mix 144 µL

PINK CAP

NEXTflex™ Adenylation Mix 216 µL

YELLOW CAP

NEXTflex™ Ligation Mix 2 (660) µL

NEXTflex™ Molecular Index Adapters (1 µM) 96 µL

GREEN CAP

NEXTflex™ qRNA-Seq™ Universal Forward Primer (10 µM) 96 µL

NEXTflex™ PCR Master Mix 576 µL

CLEAR CAP

NEXTflex™ qRNA-Seq™ Barcoded Primers 73 - 96 (10 µM) 10 µL

GRAY CAP

Nuclease-free Water (2) 1.5 mL

CLEAR CAP BOTTLE

Resuspension Buffer 15 mL


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Required Materials not Provided

• Total RNA Input: 10 ng – 1 µg total RNA for enrichment by NEXTflex™ Poly(A) Beads (Cat. # 512979, 512980, 512981). NEXTflex™ Poly(A) Beads are available separately.

• mRNA Input: ~1 ng - 100 ng isolated mRNA.

• rRNA-depleted RNA Input: ~1 ng - 100 ng. Bioo Scientific recommends Ribo-Zero™ (Epicentre) or RiboMinus™ (Life Technologies) for rRNA depletion.

• SuperScript® III Reverse Transcriptase (Life Technologies Cat # 18080-093)

• ChIP or genomic DNA Input: 500 pg - 100 ng fragmented genomic DNA or ChIP DNA

• 100% Ethanol (stored at room temperature)

• 80% Ethanol (freshly prepared)

• 2, 10, 20, 200 and 1000 µL pipettes

• RNase-free pipette tips

• Nuclease-free 1.5 mL microcentrifuge tubes

• Thin wall nuclease-free 0.5 mL microcentrifuge tubes

• 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar

• Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)

• Agencourt AMPure XP 60 mL (Beckman Coulter Genomics, Cat # A63881)

• Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar for post PCR cleanup

• Microcentrifuge

• Thermocycler

• Heat block

• Vortex



Warnings and Precautions

Bioo Scientific strongly recommends that you read the following warnings and precautions. Periodically, optimizations and revisions are made to the components and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, you may contact your local distributor or Bioo Scientific at [email protected].

• Do not use the kit past the expiration date.

• DTT in buffers may precipitate after freezing. If precipitate is seen, vortex buffer for 1-2 minutes or until the precipitate is dissolved. The performance of the buffer is not affected once precipitate is in solution.

• Ensure pipettes are properly calibrated, as library preparations are highly sensitive to pi-petting error.

• Do not heat the NEXTflex™ Molecular Index Adapters above room temperature.

• Try to maintain a laboratory temperature of 20°–25°C (68°–77°F).

• RNA sample quality may vary between preparations. High quality RNA should have a RNA Integrity Number (RIN) greater than or equal to 8, or a 28S band that is twice as intense as the 18S band of ribosomal RNA.

• ChIP DNA sample quality may vary between preparations. It is the user’s responsibility to utilize high quality ChIP DNA. ChIP DNA that is heavily nicked or damaged may cause library preparation failure. Absorbance measurements at 260 nm are commonly used to quantify DNA and 260 nm / 280 nm ratios of 1.8 - 2.0 usually indicate relatively pure ChIP DNA. Other quantification methods using fluorescent dyes may also be used. The user should be aware that contaminating DNA, nucleotides and single-stranded DNA may affect the amount of usable ChIP DNA in a sample preparation.

• DNA fragmentation methods that physically break up DNA into pieces of less than 800 bp are compatible with this kit. These methods include the AIR™ DNA Fragmentation Kit (5135-01), based on the nebulization of DNA or acoustic technologies that fragment DNA in a controlled and accurate manner. We do not recommend any enzymatic methods of fragmentation as this may introduce sequence bias into the preparation.

• Prior to library construction, ensure ChIP samples are enriched at putative targets via qPCR relative to input DNA samples.

• Vortex and micro-centrifuge each component prior to use, to ensure material has not lodged in the cap or the side of the tube.

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NEXTflex™ qRNA-Seq™ RNA Sample Preparation Flow Chart

Figure 1: Flow chart for sample preparation steps:

NEXTflex™ qRNA-SEQ™ RNA SAMPLE PREPARATION PROTOCOL

POLY A SELECTED RNA

FRAGMENTATION

FIRST STRAND cDNA SYNTHESIS

SECOND STRAND SYNTHESIS

END REPAIR

ADENYLATION

ADD MOLECULAR INDEX ADAPTERS

ADAPTER LIGATION AND LIBRARY ENRICHMENT PCR

CLUSTER GENERATION AND SEQUENCINGRead 1 Sample 1

Read 2



NEXTflex™ qChIP-Seq™ DNA Sample Preparation Flow Chart

Figure 2: Flow chart for sample preparation steps:

NEXTflex™ qChIP-SEQ™ DNA SAMPLE PREPARATION PROTOCOL

IMMUNOPRECIPITATION

30 Minutes

15 Minutes

15 Minutes

1 Hour(Optional Stop Point)

= N= A= T= Adapters with

Cluster Sequence= Molecular Labels

PROTEIN

END REPAIR

ADENYLATION

LIGATE MOLECULAR LABELS

CLUSTER GENERATION & SEQUENCING

PCR WITH BARCODED PRIMERS

Read 1 Sample 1

Read 2


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Starting Material

The NEXTflex™ qRNA-Seq™ Kit has been optimized and validated for RNA inputs using poly(A) enriched or rRNA depleted RNA (~1 ng - 100 ng) and DNA inputs using ChIP or genomic DNA (500 pg – 100 ng). Only 10 ng - 1 µg of total RNA are required if NEXTflex™ Poly(A) beads are used to enrich for mRNA*.Best results are obtained when using 50 ng or more of high quality total RNA.

For RNA inputs, follow Option 1. Bioo Scientific recommends examining total RNA integrity using an Agilent Bioanalyzer. High quality total RNA preparations should have an RNA Integri-ty Number (RIN) greater than or equal to 8. Alternatively, total RNA may be run on a 1 - 2% aga-rose gel and integrity examined by staining with ethidium bromide. High quality RNA should have a 28S band that is twice as intense as the 18S band of ribosomal RNA. Lower amounts of starting material result in higher duplication rates and other changes in sequencing data quality.

*Low RNA inputs may reduce library complexity. Read our application note about constructing high quality RNA-Seq libraries from limited amounts of total RNA. Request a PDF copy by emailing [email protected].

For DNA inputs, follow Option 2. This protocol can be used to make molecular indexed DNA libraries from as little as 500 pg of fragmented ChIP DNA. ChIP DNA sample quality may vary between preparations. It is the user’s responsibility to utilize high quality ChIP DNA. ChIP DNA that is heavily nicked or damaged may result in poor yield. Absorbance measurements at 260 nm are commonly used to quantify DNA and 260 nm / 280 nm ratios of 1.8 - 2.0 usually indicate relatively pure ChIP DNA. Other quantification methods using fluorescent dyes may also be used. The user should be aware that contaminating DNA, nucleotides and single-stranded DNA may affect the amount of usable ChIP DNA in a sample preparation.

http://www.bioongs.comhttp://www.biooscientific.com/Portals/0/White%20Papers/Constructing-High-Quality-RNA-Seq-Libraries-from-Limited-Amounts-of-Total-RNA.pdfmailto:BiooNGS%40BiooScientific.com?subject=Constructing%20High%20Quality%20RNA-Seq%20Libraries%20from%0DLimited%20Amounts%20of%20Total%20RNA%20Application%20Note


STEP A1: RNA Fragmentation

Materials

Bioo Scientific SuppliedBROWN CAP - NEXTflex™ RNA Fragmentation BufferGRAY CAP - Nuclease-free Water

User SuppliedmRNA enriched from 10 ng – 1 µg total RNA by NEXTflex™ Poly(A) Beads, or ~1 ng - 100 ng mRNA/rRNA-depleted RNA in up to 14 µL Nuclease-free Water or Elution BufferNuclease-free microcentrifuge tube or plateThermocycler or heatblock set to 95°CIce

1. For each reaction combine the following in a nuclease-free microcentrifuge tube or plate:

2. Mix well by pipetting.

3. Heat for 10 minutes at 95°C, immediately place on ice.

4. Proceed to Step B1: First Strand Synthesis.

14 µL RNA (in Nuclease-free Water or Elution Buffer)

5 µL NEXTflex™ RNA Fragmentation buffer

19 µL TOTAL

Option 1 is intended for RNA inputs.If you wish to use ChIP DNA or genomic DNA as input,

please follow Option 2.

OPTION 1 – NEXTflex™ qRNA-SEQ™


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STEP B1: First Strand Synthesis

Materials

Bioo Scientific SuppliedRED CAP - NEXTflex™ First Strand Synthesis Primer, NEXTflex™ First Strand Synthesis Buffer Mix

User SuppliedThermocycler Ice 19 µL Fragmented RNA from Step A11 µL SuperScript® III Reverse Transcriptase (Life Technologies Cat # 18080-093)

1. For each reaction, add 1 µL NEXTflex™ First Strand Synthesis Primer to the fragmented RNA (from Step A1).

2. Heat at 65°C for 5 minutes, immediately place on ice.

3. Make a First Strand Synthesis Enzyme Mix by adding 1 µL of SuperScript® III Reverse Transcriptase (Life Technologies Cat # 18080-093) per reaction to 4 µL of NEXTflex™ First Strand Synthesis Buffer Mix. Mix gently and spin down.

4. For each reaction, combine the following in a nuclease-free microcentrifuge tube or plate:


6. Incubate at:

7. Proceed to Step C1: Second Strand Synthesis.

20 µL Fragmented RNA + NEXTflex™ First Strand Synthesis Primer

5 µL First Strand Synthesis Enzyme Mix (freshly made)

25 µL TOTAL

10 min 25°C

50 min 50°C

15 min 70°C



STEP C1: Second Strand Synthesis

Materials

Bioo Scientific SuppliedBLUE CAP - NEXTflex™ Second Strand Synthesis Mix

User SuppliedThermocycler Ice 25 µL First strand synthesis product from Step B1

1. For each reaction, combine the following in a nuclease-free microcentrifuge tube or plate:


3. Incubate for 60 minutes at 16°C.

4. Proceed to Step D1: Bead Cleanup.

25 µL First strand synthesis product (from Step B1)

25 µL NEXTflex™ Second Strand Synthesis Mix

50 µL TOTAL


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STEP D1: Bead Cleanup

Materials

Bioo Scientific SuppliedCLEAR CAP BOTTLE - Resuspension Buffer

User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand 50 µL Second strand synthesis product from Step C1

1. If samples have been prepared in tubes, transfer samples to a clean 96 well PCR plate. Add 90 µL of well-mixed AMPure XP Beads to each well containing sample. Mix well by pipetting.

2. Incubate the plate for 5 minutes at room temperature.

3. Place the plate on the magnetic stand at room temperature for 5 minutes or until the supernatant appears completely clear.

4. Remove and discard the supernatant from the plate, taking care not to disturb the beads.

5. With plate on stand, add 200 µL of freshly prepared 80% ethanol to each well without disturbing the beads, and incubate the plate for at least 30 seconds at room tempera-ture. Carefully remove and discard the supernatant.

6. Repeat step 5, for a total of two ethanol washes. Ensure the ethanol has been removed.

7. Remove the plate from the magnetic stand and let dry at room temperature for 5 min-utes or until pellet appears dry.

8. Resuspend dried beads in 41 µL Resuspension Buffer. Mix well by pipetting. Ensure that the beads are completely rehydrated and resuspended.

9. Incubate resuspended beads at room temperature for 2 minutes.

10. Place the plate on the magnetic stand for 5 minutes at room temperature or until the supernatant appears completely clear.

11. Gently transfer 40 µL of the clear supernatant to a fresh well for the next step.

12. The procedure may be stopped at this point and the reactions stored at -80°C.

13. Proceed to Step E1: End Repair.



STEP E1: End Repair

Materials

Bioo Scientific SuppliedPURPLE CAP - NEXTflex™ End Repair Buffer Mix, NEXTflex™ End Repair Enzyme MixGRAY CAP - Nuclease-free water

User SuppliedThermocyclerIce40 µL Purified Second Strand Synthesis DNA from Step D1

1. For each sample, combine the following reagents on ice in a nuclease-free 96 well PCR Plate:


3. Apply adhesive PCR plate seal and incubate on a thermocycler for 30 minutes at 22°C.

4. Proceed to Step F1: Bead Cleanup.

40 µL Second Strand Synthesis DNA (from Step D1)

7 µL NEXTflex™ End Repair Buffer Mix

3 µL NEXTflex™ End Repair Enzyme Mix

50 µL TOTAL


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STEP F1: Bead Cleanup

Materials


User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand50 µL End-Repaired DNA from Step E1

1. Add 80 µL of well mixed AMPure XP Beads to each well containing sample. Mix well by pipetting.



4. Remove and discard all of the supernatant from the plate, taking care not to disturb the beads.



7. Remove the plate from the magnetic stand and let dry at room temperature for 5 min-utes or until pellet appears completely dry.






13. Proceed to Step G1: Adenylation.



STEP G1: Adenylation

Materials

Bioo Scientific SuppliedPINK CAP - NEXTflex™ Adenylation Mix

User SuppliedThermocycler 16 µL Purified End-Repaired DNA from Step F1



3. Apply adhesive PCR plate seal and incubate on a thermocycler as follows:

4. Proceed to Step H1: Adapter Ligation.

16 µL End-Repaired DNA (from Step F1)

4.5 µL NEXTflex™ Adenylation Mix

20.5 µL TOTAL

30 min 37°C

5 min 70°C


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STEP H1: Adapter Ligation

Materials

Bioo Scientific SuppliedYELLOW CAP - NEXTflex™ Ligation Mix (remove right before use and store immediately after use at -20°C), NEXTflex™ Molecular Index Adapters (for sequences labels, see pg. 33)

User SuppliedThermocycler20.5 µL 3’ Adenylated DNA from Step G1




4. Proceed to Step I1: Bead Cleanup.

20.5 µL 3’ Adenylated DNA (from Step G1)

27.5 µL NEXTflex™ Ligation Mix

2.0 µL NEXTflex™ Molecular Index Adapters

50 µL TOTAL



STEP I1: Bead Cleanup

Materials


User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand 50 µL Adapter Ligated DNA from Step H1

1. Add 40 µL of well-mixed AMPure XP Beads to each well containing sample. Mix well by pipetting.










11. Gently transfer 50 µL of the clear supernatant to a fresh well.






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24. Proceed to Step J1: PCR Amplification.



STEP J1: PCR Amplification

Materials

Bioo Scientific SuppliedGREEN CAP - NEXTflex™ PCR Master Mix, NEXTflex™ qRNA-Seq™ Universal Forward PrimerCLEAR CAP - NEXTflex™ qRNA-Seq™ Barcoded Primer

User SuppliedThermocycler96 Well PCR Plate34 µL Purified Ligated DNA from Step I1

1. For each sample, combine the following reagents on ice in the 96 well PCR plate:


3. PCR cycles:

*PCR cycles will vary depending on the amount of starting material and quality of your sample. Further optimization may be necessary. Always use the least number of cycles possible.

4. Proceed to Step K1: Bead Cleanup.

34 µL Ligated DNA (from Step I1)

12 µL NEXTflex™ PCR Master Mix

2 µL NEXTflex™ qRNA-Seq™ Universal Forward Primer

2 µL NEXTflex™ qRNA-Seq™ Barcoded Primer

50 µL TOTAL

2 min 98°C

30 sec 98°C

30 sec 65°C

60 sec 72°C

4 min 72°C

Repeat 12-15 cycles*


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STEP K1: Bead Cleanup

Materials


User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand 50 µL PCR Amplified DNA from Step J1

























23. We recommend quantifying your library with a fluorometer and checking the size us-ing an Agilent Bioanalyzer. If on the Bioanalyzer trace there are two bands, one of expected size and one of higher molecular weight, a portion of your adapter-ligated in-serts have annealed to each other forming a bubble product. This occurs due to the long adapter length and is more prevalent when there are too many PCR cycles. This type of double band will not affect your sequencing results, as the double stranded product will be denatured prior to cluster generation. As an extra verification step, a portion of your product can be denatured manually by heating the sample to 95°C for 5 minutes and then placing it on ice. The denatured product should appear as a single band on a Bioanalyzer RNA Pico 6000 Chip Kit.

24. qPCR is recommended to quantitate DNA library templates for optimal cluster density. This can be performed using any qPCR quantification kit with an Illumina-compatible qPCR primer set.

25. Non-multiplexed libraries can be normalized using Tris-HCl (10 mM), pH 8.5 with 0.1% Tween 20. For multiplexed libraries, transfer 10 µL of each normalized library for pooling in the well of a new 96 Well PCR Plate. Gently pipette the entire volume up and down 10 times.

26. Proceed to cluster generation or seal with Adhesive PCR Plate Seal and store at -20°C.


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OPTION 1 – NEXTflex™ qRNA-SEQ™ LIBRARY VALIDATION

Figure 3 : Example of mRNA library size distribution. 1 µL of the library was run on an Agilent High Sensitivity DNA chip to verify size. Using a Qubit® 2.0 Fluorometer & Qubit® dsDNA HS Assay Kit, the concentration of the library was determined to be > 10 nM.



OPTION 2 – NEXTflex™ qChIP-SEQ™

STEP A2: End Repair

Materials

Bioo Scientific SuppliedPURPLE CAP - NEXTflex™ End Repair Buffer Mix, NEXTflex™ End Repair Enzyme MixGRAY CAP - Nuclease-free Water

User Supplied500 pg - 100 ng Fragmented ChIP DNA or Fragmented genomic DNAThermocyclerIce




4. Proceed to Step B2: Bead Cleanup.

_ µL Nuclease-free Water

_ µL Fragmented ChIP DNA or Fragmented genomic DNA

7 µL NEXTflex™ End Repair Buffer Mix

3 µL NEXTflex™ End Repair Enzyme Mix

50 µL TOTAL

Option 2 is intended for ChIP DNA or genomic DNA inputs.If you wish to use RNA as input, please follow Option 1.


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STEP B2: Bead Cleanup

Materials


User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand50 µL End-Repaired DNA from Step A2: End Repair

1. Add 80 µL of well mixed AMPure XP Beads to each well containing sample. Mix well by pipetting.






7. Remove the plate from the magnetic stand and let dry at room temperature for 2 min-utes.








STEP C2: Adenylation

Materials

Bioo Scientific SuppliedPINK CAP - NEXTflex™ Adenylation Mix

User SuppliedThermocycler 16 µL Purified End-Repaired DNA from Step B2



3. Apply adhesive PCR plate seal and incubate on a thermocycler as follows:

4. Proceed to Step D2: Adapter Ligation.

16 µL End-Repaired DNA (from Step B2)

4.5 µL NEXTflex™ Adenylation Mix

20.5 µL TOTAL

30 min 37°C

5 min 70°C


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STEP D2: Adapter Ligation

Materials

Bioo Scientific SuppliedYELLOW CAP - NEXTflex™ Ligation Mix (remove right before use and store immediately after use at -20°C), NEXTflex™ Molecular Index Adapters (for sequences see pg. 33)

User SuppliedThermocycler20.5 µL 3’ Adenylated DNA from Step C2




4. Proceed to Step E2: Bead Cleanup.

20.5 µL 3’ Adenylated DNA (from Step C2)

27.5 µL NEXTflex™ Ligation Mix

2.0 µL NEXTflex™ Molecular Index Adapters

50 µL TOTAL



STEP E2: Bead Cleanup

Materials


User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand 50 µL Adapter Ligated DNA from Step D2







7. Remove the plate from the magnetic stand and let dry at room temperature for 5 min-utes or until the pellet appears dry.










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24. Proceed to Step F2: PCR Amplification.



STEP F2: PCR Amplification

Materials

Bioo Scientific SuppliedGREEN CAP - NEXTflex™ PCR Master Mix, NEXTflex™ qRNA-Seq™ Universal Forward PrimerORANGE CAP - NEXTflex™ qRNA-Seq™ Barcoded Primer

User SuppliedThermocycler96 Well PCR Plate34 µL Purified Ligated DNA from Step E2

1. For each sample, combine the following reagents on ice in the 96 well PCR plate:


3. PCR cycles:

*PCR cycles will vary depending on the amount of starting material and quality of your sample. Further optimization may be necessary. Always use the least number of cycles possible.

4. Proceed to Step G2: Bead Cleanup.

34 µL Ligated DNA (from Step E2)

12 µL NEXTflex™ PCR Master Mix

2 µL NEXTflex™ qRNA-Seq™ Universal Forward Primer

2 µL NEXTflex™ qRNA-Seq™ Barcoded Primer

50 µL TOTAL

2 min 98°C

30 sec 98°C

30 sec 65°C

60 sec 72°C

4 min 72°C

Repeat 12-28 cycles*


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STEP G2: Bead Cleanup

Materials


User SuppliedAdhesive PCR Plate Seal Agencourt AMPure XP Magnetic Beads (room temperature)80% Ethanol, freshly prepared (room temperature)Magnetic Stand 50 µL PCR Amplified DNA from Step F2

























23. We recommend quantifying your library with a fluorometer and checking the size us-ing an Agilent Bioanalyzer. If on the Bioanalyzer trace there are two bands, one of expected size and one of higher molecular weight, a portion of your adapter-ligated in-serts have annealed to each other forming a bubble product. This occurs due to the long adapter length and is more prevalent when there are too many PCR cycles. This type of double band will not affect your sequencing results, as the double stranded product will be denatured prior to cluster generation. As an extra verification step, a portion of your product can be denatured manually by heating the sample to 95°C for 5 minutes and then placing it on ice. The denatured product should appear as a single band on a Bioanalyzer RNA Pico 6000 Chip Kit.

24. qPCR is recommended to quantitate DNA library templates for optimal cluster density. This can be performed using any qPCR quantification kit with a compatible Illumina qPCR primer set.

25. Non-multiplexed libraries can be normalized using Tris-HCl (10 mM), pH 8.5 with 0.1% Tween 20. For multiplexed libraries, transfer 10 µL of each normalized library for pooling in the well of a new 96 Well PCR Plate. Gently pipette the entire volume up and down 10 times.

26. Proceed to cluster generation or seal with Adhesive PCR Plate Seal and store at -20°C.


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32

OPTION 2 – NEXTflex™ qCHIP-SEQ™ LIBRARY VALIDATION

Figure 4 : Example of Input DNA and qChIP library size distributions. 1 µL of the library was run on an Agilent High Sensitivity DNA chip to verify size. Using a Qubit® 2.0 Fluorometer & Qubit® dsDNA HS Assay Kit, the concentration of the library was determined to be > 10 nM.

High Sensitivity DNA Electropherogram

Blue - 100 pg Input DNA 18 cycle PCR productRed - 100 pg ChIP DNA 18 cycle PCR product



APPENDIX A

Molecular Index Adapters (A1-A96)

1XXXXXXXX denotes the index region of the label. The index sequences contained in each label are listed below.2XXXXXXXX denotes the complement of the index region of the label.

NEXTflex™ Sequence (5’ → 3’)

Molecular IndexAdapters

5’ GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTXXXXXXXX1T 3’ 5’ XXXXXXXX2AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC 3’

A1 AAC GCC AT A33 CAA CTG GT A65 GCC GAT TA

A2 AAG GTA CG A34 CAA GTC GT A66 GCG GTA TT

A3 AAT TCC GG A35 CAC ACA CA A67 GGA ATT GG

A4 ACA CAG AG A36 CAG TAC TG A68 GGA TAA CG

A5 ACA CTC AG A37 CAT CAG CA A69 GGC CTA AT

A6 ACA CTG TG A38 CAT CGT TC A70 GGC GTA TT

A7 ACA GGA CA A39 CCA AGG TT A71 GTC TTG TC

A8 ACC TGT AG A40 CCT AGC TT A72 GTG ATG AG

A9 ACG AAG GT A41 CGA TTA CG A73 GTG ATG TC

A10 ACG ACT TG A42 CGC CTA TT A74 GTG TAC TG

A11 ACG TCA AC A43 CGT TCC AT A75 GTG TAG TC

A12 ACG TCA TG A44 CGT TGG AT A76 GTT CAC CT

A13 ACT GTC AG A45 CTA CGT TC A77 GTT CTG CT

A14 ACT GTG AC A46 CTA CTC GT A78 GTT GTC GA

A15 AGA CAC TC A47 CTA GAG GA A79 TAC GAA CC

A16 AGA GGA GA A48 CTA GGA AG A80 TAG CAA GG

A17 AGC ATC GT A49 CTA GGT AC A81 TAG CTA GC

A18 AGC ATG GA A50 CTC AGT CT A82 TAG GTT CG

A19 AGC TAC CA A51 CTG ACT GA A83 TAT AGC GC

A20 AGC TCT AG A52 CTG AGT GT A84 TCA GGA CT

A21 AGG ACA AC A53 CTG ATG TG A85 TCC ACA TC

A22 AGG ACA TG A54 CTG TTC AC A86 TCG ACT TC

A23 AGG TTG CT A55 CTT CGT TG A87 TCG TAG GT

A24 AGT CGA GA A56 GAA CAG GT A88 TCG TCA TC

A25 AGT GCT GT A57 GAA GAC CA A89 TGA GAC TC

A26 ATA AGC GG A58 GAA GTG CA A90 TGA GAG TG

A27 ATC CAT GG A59 GAC ATG AG A91 TGA GTG AG

A28 ATC GAA CC A60 GAG AAG AG A92 TGC TTG GA

A29 ATC GCG TA A61 GAG AAG TC A93 TGG AGT AG

A30 ATC GTT GG A62 GAT CCT AG A94 TGT GTG TG

A31 CAA CGA TC A63 GAT GTC GT A95 TTC GCC TA

A32 CAA CGT TG A64 GCC GAT AT A96 TTC GTT CG


®

34

NEXTflex™ qRNA-Seq™ Primer Sequences

1XXXXXXXX denotes the index region of the primer. The index sequences contained in each primer are listed below.

Low Level Multiplexing 3 barcodes: (73, 74, 77) or (73, 76, 88)4 barcodes: (73, 74, 77, and any other) or (73, 76, 88, and any other)Please contact [email protected] for additional multiplexing guidelines.

NEXTflex™ Sequence (5’ → 3’)

qRNA-Seq™ Univer-sal Forward Primer AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGA

qRNA-Seq™Barcoded Primers

CAAGCAGAAGACGGCATACGAGATXXXXXXXX1GTGACTGGAGTTCAGACGTG

NEXTflex™ qRNA-Seq™Barcoded Primers Set A

Index 1 GTGTTCTA

Index 2 AAACATCG

Index 3 CATCAAGT

Index 4 GTCTGTCA

Index 5 AACAACCA

Index 6 CATACCAA

Index 7 TGGAACAA

Index 8 ACCTCCAA

Index 9 ACACGACC

Index 10 CTGAGCCA

Index 11 ACACAGAA

Index 12 TATCAGCA

Index 13 CTGTAGCC

Index 14 TGGCTTCA

Index 15 CCAGTTCA

Index 16 AGATGTAC

Index 17 ACAGCAGA

Index 18 ACAAGCTA

Index 19 ACGCTCGA

Index 20 CACTTCGA

Index 21 AAGACGGA

Index 22 AACGCTTA

Index 23 GAATCTGA

Index 24 ATCCTGTA

NEXTflex™ qRNA-Seq™Barcoded Primers Set B

Index 25 AGTACAAG

Index 26 ACCACTGT

Index 27 AACTCACC

Index 28 CGCATACA

Index 29 AGGCTAAC

Index 30 GCCAAGAC

Index 31 ATGCCTAA

Index 32 GGTGCGAA

Index 33 CAATGGAA

Index 34 ATCATTCC

Index 35 CAAGACTA

Index 36 AACCGAGA

Index 37 AAGAGATC

Index 38 CAAGGAGC

Index 39 CCGTGAGA

Index 40 CCGAAGTA

Index 41 ACAGATTC

Index 42 GACTAGTA

Index 43 CGACTGGA

Index 44 CGGATTGC

Index 45 ACATTGGC

Index 46 AATGTTGC

Index 47 CAACCACA

Index 48 CCGACAAC

NEXTflex™ qRNA-Seq™Barcoded Primers Set C

Index 49 AACGTGAT

Index 50 GGAGAACA

Index 51 AAGGACAC

Index 52 GTACGCAA

Index 53 AGAGTCAA

Index 54 TTCACGCA

Index 55 CACCTTAC

Index 56 GAGCTGAA

Index 57 GCCACATA

Index 58 AGCACCTC

Index 59 GCTAACGA

Index 60 AGTCACTA

Index 61 GATGAATC

Index 62 TGAAGAGA

Index 63 CGCTGATC

Index 64 GAGTTAGC

Index 65 ATTGGCTC

Index 66 CGAACTTA

Index 67 AATCCGTC

Index 68 ATAGCGAC

Index 69 TAGGATGA

Index 70 CAGCGTTA

Index 71 GCTCGGTA

Index 72 CGACACAC

NEXTflex™ qRNA-Seq™Barcoded Primers Set D

Index 73 CAGATCTG

Index 74 GATAGACA

Index 75 AAGGTACA

Index 76 CCTAATCC

Index 77 AGATCGCA

Index 78 ACGTATCA

Index 79 AGCAGGAA

Index 80 ACTATGCA

Index 81 GCGAGTAA

Index 82 AGTGGTCA

Index 83 CCATCCTC

Index 84 CTGGCATA

Index 85 AGCCATGC

Index 86 CCTCTATC

Index 87 TCCGTCTA

Index 88 GTCGTAGA

Index 89 GAACAGGC

Index 90 CTCAATGA

Index 91 CCTCCTGA

Index 92 ATTGAGGA

Index 93 GACAGTGC

Index 94 CTAAGGTC

Index 95 TGGTGGTA

Index 96 TCTTCACA

http://www.bioongs.commailto:BiooNGS%40biooscientific.com?subject=NEXTflex%20qRNA%20Low%20Level%20Multiplexing%20Guidelines


RELATED PRODUCTS

Illumina Compatible RNA NGS Kits and Adapters

Catalog # Product

5138-01 NEXTflex™ Rapid RNA-Seq Kit (8 reactions)

5138-02 NEXTflex™ Rapid RNA-Seq Kit (48 reactions)

5138-07 NEXTflex™ Rapid Directional RNA-Seq Kit (8 reactions)

5138-08 NEXTflex™ Rapid Directional RNA-Seq Kit (48 reactions)

512911 NEXTflex™ RNA-Seq Barcodes –6

512912 NEXTflex™ RNA-Seq Barcodes – 12



512916 NEXTflex-96™ RNA-Seq Barcodes

5130-01 NEXTflex™ qRNA-Seq™ Kit 4 barcodes (8 reactions)

5130-02 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set A (48 reactions)

5130-03 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set B (48 reactions)

5130-04 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set C (48 reactions)

5130-05 NEXTflex™ qRNA-Seq™ Kit 24 barcodes - Set D (48 reactions)

5130-01D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 4 barcodes (8 reactions)

5130-02D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set A (48 reactions)

5130-03D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set B (48 reactions)

5130-04D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set C (48 reactions)

5130-05D NEXTflex™ Rapid Directional qRNA-Seq™ Kit 24 barcodes - Set D (48 reactions)

5132-01 NEXTflex™ Small RNA Sequencing Kit (24 reactions)

5132-02 NEXTflex™ Small RNA Sequencing Kit (48 reactions)

5132-03 NEXTflex™ Small RNA Sequencing Kit v2 (24 reactions)

5132-04 NEXTflex™ Small RNA Sequencing Kit v2 (48 reactions)

513305 NEXTlfex™ Small RNA Barcode Primers -12 (Set A)

513306 NEXTlfex™ Small RNA Barcode Primers -12 (Set B)

513307 NEXTlfex™ Small RNA Barcode Primers -12 (Set C)

513308 NEXTlfex™ Small RNA Barcode Primers -12 (Set D)

512979 NEXTflex™ Poly(A) Beads (8 reactions)



http://www.bioongs.comhttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-rapid-rna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-rapid-rna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-stranded-rna-seq-library-prep-kits/nextflex-rapid-directional-rna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-stranded-rna-seq-library-prep-kits/nextflex-rapid-directional-rna-seq-kithttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2RNA-SeqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2RNA-SeqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2RNA-SeqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2RNA-SeqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex-96%E2%84%A2RNA-SeqBarcodes.aspxhttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-rna-seq-library-prep-kits/nextflex-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-rapid-directional-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-rapid-directional-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-rapid-directional-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-rapid-directional-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-rapid-directional-qrna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/nextflex-illumina-small-rna-seq-library-prep-kitshttp://www.biooscientific.com/next-gen-sequencing/nextflex-illumina-small-rna-seq-library-prep-kitshttp://www.biooscientific.com/next-gen-sequencing/nextflex-illumina-small-rna-seq-library-prep-kit-v2http://www.biooscientific.com/next-gen-sequencing/nextflex-illumina-small-rna-seq-library-prep-kit-v2http://www.biooscientific.com/next-gen-sequencing/illumina-small-rna-seq-adapters/nextflex-small-rna-barcode-primershttp://www.biooscientific.com/next-gen-sequencing/illumina-small-rna-seq-adapters/nextflex-small-rna-barcode-primershttp://www.biooscientific.com/next-gen-sequencing/illumina-small-rna-seq-adapters/nextflex-small-rna-barcode-primershttp://www.biooscientific.com/next-gen-sequencing/illumina-small-rna-seq-adapters/nextflex-small-rna-barcode-primershttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2Poly(A)Beads.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2Poly(A)Beads.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/RNA-Seq/NEXTflex%E2%84%A2Poly(A)Beads.aspx

®

36

Illumina Compatible DNA NGS Kits and Adapters

Catalog # Product

4201-01 NEXTflex™ 16S V4 Amplicon-Seq Kit – 4

4201-02 NEXTflex™ 16S V4 Amplicon-Seq kit – 12






4202-01 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 4



4202-04 NEXTlfex™ 16S V1-V3 Amplicon-Seq Kit - 1-96




5140-01 NEXTflex™ DNA Sequencing Kit (8 reactions)

5140-02 NEXTflex™ DNA Sequencing Kit (48 reactions)

5144-01 NEXTflex™ Rapid DNA-Seq Kit (8 reactions)

5144-02 NEXTflex™ Rapid DNA-Seq Kit (48 reactions)

5150-01 NEXTflex™ Cell Free DNA-Seq Kit (8 reactions)

5150-02 NEXTflex™ Cell Free DNA-Seq Kit (48 reactions)

514101 NEXTflex™ DNA Barcodes – 6




514105 NEXTflex-96™ DNA Barcodes (Plate Format)

514106 NEXTflex-96™ DNA Barcodes (Tube Format)

514160 NEXTflex™ Dual-Indexed DNA Barcodes (1-96)

514161 NEXTflex™ Dual-Indexed DNA Barcodes (97-192)

5119-01 NEXTflex™ Bisulfite-Seq kit (8 reactions)

5119-02 NEXTflex™ Bisulfite-Seq kit (48 reactions)

511911 NEXTflex™ Bisulfite-Seq Barcodes – 6

511912 NEXTflex™ Bisulfite-Seq Barcodes – 12

511913 NEXTflex™ Bisulfite-Seq Barcodes - 24

5118-01 NEXTflex™ Methyl-Seq 1 Kit (8 reactions)

5118-02 NEXTflex™ Methyl-Seq 1 Kit (48 reactions)

http://www.bioongs.comhttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v4-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-16s-v1-v3-amplicon-seq-kithttp://www.biooscientific.com/products-and-services/next-gen-sequencing/illumina-library-prep-kits/dna-seq/nextflex-dna-sequencing-kithttp://www.biooscientific.com/products-and-services/next-gen-sequencing/illumina-library-prep-kits/dna-seq/nextflex-dna-sequencing-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kits/nextflex-rapid-dna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kits/nextflex-rapid-dna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-cell-free-dna-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-cell-free-dna-seq-kithttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2DNABarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2DNABarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2DNABarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2DNABarcodes.aspxhttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-dna-barcodeshttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex-96%E2%84%A2DNABarcodes.aspxhttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-dual-index-dna-barcodeshttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-dual-index-dna-barcodeshttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/methyl-seq/nextflex-bisulfite-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/methyl-seq/nextflex-bisulfite-seq-kithttp://www.biooscientific.com/next-gen-sequencing/methylated-illumina-adapters/nextflex-bisulfite-seq-barcodeshttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/Methyl-Seq/NEXTflex%E2%84%A2Bisulfite-SeqBarcodes.aspxhttp://www.biooscientific.com/next-gen-sequencing/methylated-illumina-adapters/nextflex-bisulfite-seq-barcodeshttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/Methyl-Seq/NEXTflex%E2%84%A2Bisulfite-SeqBarcodes.aspxhttp://www.biooscientific.com/next-gen-sequencing/methylated-illumina-adapters/nextflex-bisulfite-seq-barcodeshttp://www.biooscientific.com/next-gen-sequencing/methylated-illumina-adapters/nextflex-bisulfite-seq-barcodeshttp://www.biooscientific.com/products-and-services/next-gen-sequencing/illumina-library-prep-kits/methyl-seq/nextflex-methyl-seq-1-kithttp://www.biooscientific.com/products-and-services/next-gen-sequencing/illumina-library-prep-kits/methyl-seq/nextflex-methyl-seq-1-kit


511921 NEXTflex™ Msp 1 (8 reactions)

511922 NEXTflex™ Msp 1 (48 reactions)

5143-01 NEXTflex™ ChIP-Seq Kit (8 reactions)

5143-02 NEXTflex™ ChIP-Seq Kit (48 reactions)

514120 NEXTflex™ ChIP-Seq Barcodes – 6




514124 NEXTflex-96™ ChIP-Seq Barcodes

5140-51 NEXTflex™ Pre-Capture Combo Kit (6 barcodes)





514131 NEXTflex™ DNA Barcode Blockers - 6 for SeqCap





5142-01 NEXTflex™ PCR-Free DNA Sequencing Kit (8 reactions)

5142-02 NEXTflex™ PCR-Free DNA Sequencing Kit (48 reactions)

514110 NEXTflex™ PCR-Free Barcodes – 6




DNA Fragmentation

Catalog # Product

5135-01 AIR™ DNA Fragmentation Kit (10 reactions)

5135-02 AIR™ DNA Fragmentation Kit (40 reactions)

http://www.bioongs.comhttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/bisulfite-seq/nextflex-msp1-restriction-enzymehttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/bisulfite-seq/nextflex-msp1-restriction-enzymehttp://www.biooscientific.com/next-gen-sequencing/illumina-chip-seq-library-prep-kit/nextflex-chip-seq-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-chip-seq-library-prep-kit/nextflex-chip-seq-kithttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2ChIP-seqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2ChIP-seqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2ChIP-seqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex%E2%84%A2ChIP-seqBarcodes.aspxhttp://www.biooscientific.com/ProductsServices/NextGenSequencing/Illumina-Compatible/DNA-Seq/NEXTflex-96%E2%84%A2ChIP-SeqBarcodes.aspxhttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kit/nextflex-pre-capture-combo-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kit/nextflex-pre-capture-combo-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kit/nextflex-pre-capture-combo-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kit/nextflex-pre-capture-combo-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-dna-library-prep-kit/nextflex-pre-capture-combo-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/pre-capture-combo/nextflex-dna-barcode-blockershttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/pre-capture-combo/nextflex-dna-barcode-blockershttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/pre-capture-combo/nextflex-dna-barcode-blockershttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/pre-capture-combo/nextflex-dna-barcode-blockershttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/pre-capture-combo/nextflex-dna-barcode-blockershttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-pcr-free-dna-sequencing-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-library-prep-kits/nextflex-pcr-free-dna-sequencing-kithttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-pcr-free-barcodeshttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-pcr-free-barcodeshttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-pcr-free-barcodeshttp://www.biooscientific.com/next-gen-sequencing/illumina-adapters/dna-seq/nextflex-pcr-free-barcodeshttp://www.biooscientific.com/products-and-services/next-gen-sequencing/illumina-library-prep-kits/dna-seq/air-dna-fragmentation-kithttp://www.biooscientific.com/products-and-services/next-gen-sequencing/illumina-library-prep-kits/dna-seq/air-dna-fragmentation-kit

®

38

NOTES


®

40


�is product is for research use only.

�is manual is proprietary to Bioo Scienti�c Corp., and intended only for customer use in connection with the product(s) described herein and for no other purpose. �is document and its contents shall not be used or distributed for any other purpose without the prior written consent of Bioo Scienti�c. Periodic optimizations and revisions are made to kit components and manu-als. Follow the protocol included with the kit.

Bioo Scienti�c makes no warranty of any kind, either expressed or implied, except that the materials from which its products are made are of standard quality. �ere is no warranty of merchantability for this product, or of the �tness of the product for any purpose. Bioo Scienti�c shall not be liable for any damages, including special or consequential damages, or expense arising directly or indirectly from the use of this product.

Bioo Scienti�c, NEXT�ex, AIR, �e NGS Experts, qRNA, and NanoQ are trademarks or registered trademarks of Bioo Scienti�c. All other brands and names contained herein are the property of their respective owners.

WE WANT TO HEAR FROM YOU!Your feedback is important to us. Tell us what you

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®

®

Bioo Scienti�c Corporation · 7050 Burleson Road, Austin, Texas 78744 · BiooScientific.comP: 1.888.208.2246 · F: 512.707.8122 · Bioo Research Products Group · [email protected]

Made in the USA

THE NGS EXPERTS™

FOR REFERENCE PURPOSESThis manual is for Reference Purposes Only. DO NOT use

this protocol to run your assays. Periodically, optimizations

and revisions are made to the kit and protocol, so it is

important to always use the protocol included with the kit.

GENERAL INFORMATIONProduct OverviewRevision HistoryContents, Storage and Shelf LifeRequired Materials not ProvidedWarnings and Precautions

NEXTflex™ qRNA-SEQ™ RNA SAMPLE PREPARATION PROTOCOLNEXTflex™ qRNA-Seq™ RNA Sample Preparation Flow Chart

NEXTflex™ qChIP-SEQ™ DNA SAMPLE PREPARATION PROTOCOLNEXTflex™ qChIP-Seq™ DNA Sample Preparation Flow ChartStarting Material

OPTION 1 – NEXTflex™ qRNA-SEQ™STEP A1: RNA FragmentationSTEP B1: First Strand SynthesisSTEP C1: Second Strand SynthesisSTEP D1: Bead CleanupSTEP E1: End RepairSTEP F1: Bead CleanupSTEP G1: AdenylationSTEP H1: Adapter LigationSTEP I1: Bead CleanupSTEP J1: PCR AmplificationSTEP K1: Bead Cleanup

OPTION 1 – NEXTflex™ qRNA-SEQ™ LIBRARY VALIDATION OPTION 2 – NEXTflex™ qChIP-SEQ™STEP A2: End RepairSTEP B2: Bead CleanupSTEP C2: AdenylationSTEP D2: Adapter LigationSTEP E2: Bead CleanupSTEP F2: PCR AmplificationSTEP G2: Bead Cleanup

OPTION 2 – NEXTflex™ qCHIP-SEQ™ LIBRARY VALIDATIONAPPENDIX AMolecular Index Adapters (A1-A96)NEXTflex™ qRNA-Seq™ Primer SequencesLow Level Multiplexing

RELATED PRODUCTSNOTESGENERAL INFORMATIONProduct OverviewRevision HistoryContents, Storage and Shelf LifeRequired Materials not ProvidedWarnings and Precautions
























RELATED PRODUCTSNOTES

for reference purposes · 2020. 11. 24. · bioo scienti˜c corporation · 7050 burleson road,...

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