January 2013 TrainingChIP-Seq & Stem Cell Dye

Agenda

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• Active Motif is introducing 2 new products in our next issue of Motifvations which will reach customers the first week of February 2013

Product Format Catalog No.

ChIP-IT® ChIP-Seq 10 libraries 53041

Product Format Catalog No.

Stem Cell CDy1 Dye 50 µl 14001

ChIP-Seq

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• What is ChIP-Seq?– ChIP-Seq is a powerful technique that combines

chromatin immunoprecipitation (ChIP) with Next Generation sequencing for genome-wide analysis

– The ChIP method enriches for protein/DNA complexes of interest. The enriched DNA is then modified with oligonucleotide adapters to generate a sequencing library.

• Why perform ChIP-Seq?– ChIP-seq can provide insights into gene regulation by

identifying global DNA binding sites for proteins of interest

ChIP-Seq Workflow

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• The ChIP-IT® ChIP-Seq Kit provides optimized reagents and protocols to perform the complete ChIP-Seq workflow:– Chromatin preparation– Chromatin IP– DNA purification– ChIP DNA validation– Sequencing Library

Construction

ChIP-IT® ChIP-Seq

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• The ChIP-IT® ChIP-Seq Kit contains 3 modules:

• Chromatin Preparation and Immunoprecipitation– Same reagents as the ChIP-IT High Sensitivity Kit– Enough material for 16 chromatin preps and 16 ChIPs

• ChIP-IT qPCR Analysis Kit– Complete Kit with positive and negative control qPCR

primers to validate the success of the ChIP reaction

• Sequencing Library Construction Reagents– Contains the enzymes and protocols needed to generate a

sequencing library for use with the Illumina® platforms– Enough material is supplied to construct 10 libraries

Major points of interest

• ChIP-IT ChIP-Seq is recommended for use with at least 30 µg chromatin (4.5 million cell equivalents)– 10 µg chromatin (1.5 million cell equivalents) can be used

with highly abundant histone targets– For good ChIP-Seq results we do not recommend the use

of low cell numbers as discussed in the High Sensitivity Kit

• Use of the ChIP-IT qPCR Analysis Kit will provide guidelines for ChIP DNA quality prior to sequencing– If customers do not use our qPCR analysis kit, the

recommendation thresholds do not apply

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Major points of interest

• The Sequencing Library Construction Protocol is designed for use with Next Generation sequencing using the Illumina® platforms– Genome Analyzer II– HiSeq– MiSeq

• Adapters and amplification primers must be obtained from Illumina for single end, paired end or barcoded library generation– Alternate supplier is Bioo Scientific, NEXTflex ChIP-Seq

Barcodes (Cat 514120)

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ChIP-IT® ChIP-Seq

• ChIP-IT ChIP-Seq Kit Advantages:– Optimized reagents and the use of our antibody blocker

reduce background levels caused by non-specific binding events. This leads to better enrichment, and therefore larger ChIP-Seq peaks for more accurate peak calling since a larger percentage of the sequence reads are mapped to binding sites rather than background regions

– The ChIP filtration columns offer faster, easier wash steps. The filtration columns retain the Protein G agarose beads preventing sample loss for better consistency in results

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ChIP-IT® ChIP-Seq

• ChIP-IT ChIP-Seq Kit Advantages (cont.):– The protocol has been validated for use with low

abundance transcription factor antibodies or antibodies with low binding affinities

– The included ChIP-IT qPCR analysis kit provides guidelines to confirm the quality of the ChIP-enriched DNA prior to sequencing so time and money is not wasted on expensive sequencing of poor quality ChIP DNA. The qPCR Analysis Kit gives customers confidence in successful ChIP-Seq results

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ChIP-IT® ChIP-Seq

• ChIP-IT ChIP-Seq Kit Advantages (cont.):– The kit includes optimized protocols and reagent for

successful sequencing library preparation of single end, paired end or barcoded libraries

– We use AMPure® XP beads for purification of the final library which provides a higher purity library than Qiagen column purification

– The ChIP-IT ChIP-Seq Kit has been validated to work on Illumina’s Genome Analyzer II, HiSeq and MiSeq platforms.

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Competition

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AM Millipore Diagenode Diagenode Illumina

Format 16 ChIPs10 libraries

22 ChIPs10 libraries

10 ChIPs 10 ChIPs12 libraries

10 libraries

Price $815 $719 $520 $2195 $2300

Included • Agarose Prot. G

• ChIP filter columns

• Pos/Neg primers & guidelines

• Enzymes for seq.

• Magnetic Prot. A/G

• Pos/Neg control Abs & primers

• Enzymes for seq.

• Magnetic Prot A

• Pos/Neg control Ab & primers

• Protocol for seq.

• Magnetic Prot A

• Pos/Neg control Ab & primers

• Index adapters

• Enzymes for seq.

• Adapters• Enzymes

for seq.

Not Included

• Adapters• Qiagen kits• AMPure

beads

• Adapters• Qiagen kits

• Enzymes for library generation

• Qiagen kits

• AMPure beads

• ChIP reagents

• Qiagen kits

Sales Strategy

• ChIP-IT ChIP-Seq– Customers intending to perform ChIP-Seq– Will invest money into sequencing and data analysis and

want to ensure quality sequencing reads by using a kit with reagents that reduce non-specific DNA & a strategy to validate ChIP DNA quality

– Ideally have 4.5 M cell equivalents to use per ChIP– Price savings by purchasing reagents together:

» ChIP-IT High Sensitivity $440» ChIP-IT qPCR Analysis $ 85» Sequencing Enzymes $410» TOTAL $935» ChIP-Seq Kit Price $815

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Sales Strategy

• ChIP-IT High Sensitivity– Designed for ChIP from limited sample amounts,

tissues, or for low abundance targets– While ChIP DNA from this kit has been validated to

work in ChIP-Seq, the focus of the kit was on reducing the background for better ChIP enrichment of the protein of interest

• ChIP-IT Express– Designed as fast, easy ChIP protocol when working

with >100,000 cells– Ideal for analysis of robust gene targets

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Sales Strategy

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• Cross-selling opportunities:– EpiShear sonicator– Cooled sonication platform– Dounce homogenizer– ChIP-seq validated antibodies– ChIP qPCR primer sets

Stem Cell CDy1 Dye

• Stem cells – pluripotent cells capable of self-renewal. These cells are important research tools in understanding development, aging, regeneration and therapeutics– Embryonic Stem Cells (ESCs) – are usually grown

on a feeder layer of MEF cells. Stem cells must then be identified and isolated from MEFs

– Induced Pluripotent Stem Cells (iPSCs) – are differentiated cells that are reprogrammed to a pluripotent state. Pluripotent cells are then identified and isolated from non-reprogrammed cells

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Stem Cell CDy1 Dye

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• Active Motif has obtained a stem cell specific dye from researchers in Singapore

• The CDy1 dye specifically stains pluripotent cells (mESC, hESC and iPSC)– This allows ESCs to be identified from feeder cells &

iPSCs to be identified from non-reprogrammed cells– This dye will also allow researchers to test stem cell

cultures to ensure that the stem cells have not begun to differentiate

Stem Cell CDy1 Dye

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• How are stem cells normally identified?– Alkaline phosphatase (AP) assay – used for

phenotypic assessment of ESC differentiation by measuring AP activity. ESC express high levels of AP. This usually involves fixation or sacrifice of the cells.

– Aldefluor assay – screens for stem & progenitor cells that are expressing high levels of aldehyde dehydrogenase in hematopoietic cells (other cells are possible). The cells are sorted by flow cytometry.

– Immunostaining – uses antibodies to surface markers or TF markers to identify pluripotency. This involves fixation of the cells.

Stem Cell CDy1 Dye

• How does the Stem Cell CDy1 work?– CDy1 dye is added to the culture media of live cells– Incubation for 1 hour at 37˚C– Cells are washed and prepared for imaging by

fluorescent microscopy or FACS analysis– Additional experiments or immunostaining for other

pluripotent markers can be performed after CDy1 staining

• Unknowns– The exact mechanism of the dye staining– Optimizations may be required for each cell type

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Stem Cell CDy1 Dye

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• CDy1 Stain Advantages:– The dye works on live cells so there is no need for

fixation or cell sacrifice– The stain can be visualized by fluorescent

microscopy or flow cytometry– The dye works with normal fluorescent filter sets

(TRITC / Cy3 and PE-Texas-Red)– The dye does not affect the stem cells ability to

differentiate. The dye will disappear upon differentiation

– Ease-of-use enables higher throughput screening of pluripotent stem cells

Stem Cell CDy1 Dye

• Current plan of action:– Use of the CDy1 dye has been published with detailed

protocols and staining data» Kang et al. (2011) Nature Protocols 6 (7): 1044-1052

– We will utilize this publication and distribute it through the sales force to interested labs

– Details from the publication methods will be reproduced in our documents and protocols

– Beta-test samples are already in customer hands– We will compile customer information to further optimize

our protocols

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Competition

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Method Company Product Format Price

CDy1 Stain Active Motif CDy1 Dye 50 µl $285

Alkaline Phosphatase

Millipore Quantitative AP Kit 100 tests $227

Alkaline Phosphatase

Invitrogen AP Live Stain 50 µl $212

Alkaline Phosphatase

Invitrogen ELF 97 Phosphatase Kit

1 kit $278

Aldefluor Stem Cell Tech

Aldefluor Kit 1 kit $541

Immunostaining CST StemLight 40 µl per Ab $256-396

Immunostaining Millipore ESC marker 100 µl per Ab $682

Flow cytometry Millipore FlowCellect 100 tests $750

Sales Strategy

• Position the CDy1 dye against the AP kits, but due to the added advantage of working on live cells, we are at a higher price point

• CDy1 is less expensive than immunostaining kits and the two can be used together on a single sample set

• The dye can also be used for FACS analysis, but as we have limited protocols for this application we may create a second pack size and price point for the dye in the future for use with FACS

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