supplementary fig1.1 for mp - media.nature.com · ncp mir-484p ncp mir-484p nci mir-484i nci...
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Supplementary Figure 1Establishment of the gain- and loss-of-function experiments and cell survival assays.
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NCP miR-484P NCP
miR-484P NCI
miR-484I NCI
miR-484I
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cTuj1(-) Tuj1(+)
apoptotic live
Tuj1
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j1G
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apoptotic live
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WB:anti-mycAntibody
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scr sh10.0
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H19
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Supplementary Figure 2Assessment of the PCDH19 antibody and Pcdh19 shRNAvector.
WB:anti-PCDH19Antibody
Supplementary Table 1 Clinical manifestation data obtained from the DECIPHER/ISCA databases and reported papers.
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Supplementary Materials and Methods
Plasmids
Full-length cDNA fragments encoding mouse Nde1 (NM_023317.2; 313-1513, Primers:
forward, 5'- TAT CTC GAG ATG GCA TCA ATG CAG AAG CTG ATC TCA GAG GAG
GAC CTG GAG GAC TCG GGA AAG ACC TT -3', reverse, 5'- CAG GAC TAT GCG GCC
GCC TAA AGC AAA AGC TTG ACC A -3'), mouse Fopnl (NM_025345.2; 1-878, Primers:
forward, 5'- TAT CTC GAG ATG GCA TCA ATG CAG AAG CTG ATC TCA GAG GAG
GAC CTG GCA TCA ATG CAG AAG CT -3', reverse, 5'- CAG GAC TAT GCG GCC GCT
ACA TGA TGT TGC ACT GGG T), and mouse Pcdh19 (NM_001105245.1, Primers: forward,
5'- TTA GCG GCC GCC GCG CAG CCA TGG AGT CTC TCC TGC TGC CG -3', reverse, 5'-
TTA GCG GCC GCT TAC AGG TCC TCC TCT GAG ATC AGC TTC TGC TCG CCG TTG
AGA ACG ATA TCC TTC AGA C -3') tagged with an N-terminal Myc epitope were cloned
into a pCAGIG vector (Addgene) by restriction enzyme digestion. Elongation factor 1α (EF1α)
promoter-driven nuclear enhanced green fluorescent protein (EGFP) reporter expression
plasmid was used as an indication of transfected cells.17 Genome sequence oligos encoding
3'-untranslated region (UTR) of Emx1-1 (sense, 5'- AAA CTA GCG GCC GCG CCT CGA
GTC ACG GCC CCA CAG GGC TTG GAG CCT GGG GCC ATC ACT T -3', anti-sense, 5'-
CTA GAA GTG ATG GCC CCA GGC TCC AAG CCC TGT GGG GCC GTG ACT CGA
GGC GCG GCC GCT AGT TT-3'), Emx1-2 (sense, 5'- AAA CTA GCG GCC GCG CCT GGG
ACT GCT CAG CCT TCT GGT GGA GAG CCT GCT GCT TGG GTG T -3', anti-sense, 5'-
CTA GAC ACC CAA GCA GCA GGC TCT CCA CCA GAA GGC TGA GCA GTC CCA
GGC GCG GCC GCT AGT TT-3'), Emx1-3 (sense, 5'- AAA CTA GCG GCC GCA TGA CGT
AGC TAT CCA TCT TCA ACC ATA GAG CCT GGG CTG TTG GGT T -3', anti-sense, 5'-
CTA GAA CCC AAC AGC CCA GGC TCT ATG GTT GAA GAT GGA TAG CTA CGT
CAT GCG GCC GCT AGT TT-3'), Emx1-4 (sense, 5'- AAA CTA GCG GCC GCC TGG TCA
GCA AGA GCG CTC AGG GCC CAG GAG CCT GAA GGG AAG CCT T -3', anti-sense, 5'-
CTA GAA GGC TTC CCT TCA GGC TCC TGG GCC CTG AGC GCT CTT GCT GAC CAG
GCG GCC GCT AGT TT-3'), Fgf1 (sense, 5'- AAA CTA GCG GCC GCA TTG GCT GCG
CTA ACC CTC AGG CCA CAG AGC CTG AAT TTG TAA GCA T -3', anti-sense, 5'- CTA
GAT GCT TAC AAA TTC AGG CTC TGT GGC CTG AGG GTT AGC GCA GCC AAT
GCG GCC GCT AGT TT-3'), Strn (sense, 5'- AAA CTA GCG GCC GCG GTT GCT GCA
AGC TCA GCC AAA CCT GTG AGC CTG AAA CTT TGA GAT T -3', anti-sense, 5'- CTA
GAA TCT CAA AGT TTC AGG CTC ACA GGT TTG GCT GAG CTT GCA GCA ACC
GCG GCC GCT AGT TT -3'), Ltbp2 (sense, 5'- AAA CTA GCG GCC GCT AAG AAA TAC
AAA GGA CGA GGA ACA GTG AGC CTG AGA AAG TAC GGC T -3', anti-sense, 5'- CTA
GAG CCG TAC TTT CTC AGG CTC ACT GTT CCT CGT CCT TTG TAT TTC TTA GCG
GCC GCT AGT TT -3'), TGFRB3 (sense, 5'- AAA CTA GCG GCC GCA ATA CTC TAT TCT
GGT TTT TCC TGA GTG AGC CTG ATT TTG TTC CTG T -3', anti-sense, 5'- CTA GAC
AGG AAC AAA ATC AGG CTC ACT CAG GAA AAA CCA GAA TAG AGT ATT GCG
GCC GCT AGT TT-3'), Trim71 (sense, 5'- AAA CTA GCG GCC GCG AAT GTA ATT CTC
CTC TGT GCA GGG CTT GAG CCT GTG AAG TGA TAA T -3', anti-sense, 5'- CTA GAT
TAT CAC TTC ACA GGC TCA AGC CCT GCA CAG AGG AGA ATT ACA TTC GCG
GCC GCT AGT TT-3'), Hivep2 (sense, 5'- AAA CTA GCG GCC GCC AAT TAG GTA AAT
TAA CAA GTC ATC TTG AGC CTG ACC AAA ACA AAA T -3', anti-sense, 5'- CTA GAT
TTT GTT TTG GTC AGG CTC AAG ATG ACT TGT TAA TTT ACC TAA TTG GCG GCC
GCT AGT TT-3'), Htt (sense, 5'- CTA GAG GAC CCA GGA CTC AGG CTC AAC CCC
AAC ATA GTG CAT GTT CCC TGC GCG GCC GCT AGT TT -3', anti-sense, 5'- AAA CTA
GCG GCC GCC TCT CAA CCA GCT GGA GAG ATA CCT CAG AGC CTG ACC ATA
GTT CAT T-3'), Atp7B (sense, 5'- AAA CTA GCG GCC GCC TCT CAA CCA GCT GGA
GAG ATA CCT CAG AGC CTG ACC ATA GTT CAT T -3', anti-sense, 5'- CTA GAA TGA
ACT ATG GTC AGG CTC TGA GGT ATC TCT CCA GCT GGT TGA GAG GCG GCC
GCT AGT TT-3'), Ptch19 (sense, 5'- AAA CTA GCG GCC GCG CCT TCG CTC CTC TAG
TGA GGG GAA TGA GCC TGA TGA CAC TAT TAC T -3', anti-sense, 5'- CTA GAG TAA
TAG TGT CAT CAG GCT CAT TCC CCT CAC TAG AGG AGC GAA GGC GCG GCC
GCT AGT TT -3'), Prrt2 (sense, 5'- AAA CTA GCG GCC GCC CTG GCC CCC ATC TCT
AGG GAC CAA GGG AGC CTG AGC GGC CTT GTT T -3', anti-sense, 5'- CTA GAA ACA
AGG CCG CTC AGG CTC CCT TGG TCC CTA GAG ATG GGG GCC AGG GCG GCC
GCT AGT TT -3'), Hspg2 (sense, 5'- AAA CTA GCG GCC GCG GGA TGA TGA TAA CCC
TGC CCA TCT GGG AGC CTG AGG ACC TGA CTA T -3', anti-sense, 5'- CTA GAT AGT
CAG GTC CTC AGG CTC CCA GAT GGG CAG GGT TAT CAT CAT CCC GCG GCC
GCT AGT TT -3'), Ptpre (sense, 5'- AAA CTA GCG GCC GCT TAT GTG TTT TTA AAA
CCT TTT TAA ATG AGC CTG ATA CCT GTG TTT T -3', anti-sense, 5'- CTA GAA AAC
ACA GGT ATC AGG CTC ATT TAA AAA GGT TTT AAA AAC ACA TAA GCG GCC
GCT AGT TT -3'), Pik3cd (sense, 5'- AAA CTA GCG GCC GCA TGT CCA GTG GTC ACC
AAA GGG ACC CCA GAG CCT GAA CTG TCC CTC T -3', anti-sense, 5'- CTA GAG AGG
GAC AGT TCA GGC TCT GGG GTC CCT TTG GTG ACC ACT GGA CAT GCG GCC
GCT AGT TT -3'), and Znf667 (sense, 5'- AAA CTA GCG GCC GCT AGA GTC CAC TTT
TCT GGT GGA GCT TTG AGC CTG AGT TGA GGT CCC T -3', anti-sense, 5'- CTA GAG
GGA CCT CAA CTC AGG CTC AAA GCT CCA CCA GAA AAG TGG ACT CTA GCG
GCC GCT AGT TT -3') were annealed and cloned into pmirGLO Dual-Luciferase miRNA
Target Expression Vector (Promega) Oligos containing mismatch sequence (sense, 5'- AAA
CTA GCG GCC GCG CCT TCG CTC CTC TAG TGA GGG GAA TTC ACT GAT GAC ACT
ATT ACT -3', anti-sense, 5'- CTA GAG TAA TAG TGT CAT CAG TGA ATT CCC CTC
ACT AGA GGA GCG AAG GCG CGG CCG CTA GTT T -3'), which was also annealed and
cloned into the same vector. Ptch19 containing mismatch sequence (sense, 5'- AAA CTA GCG
GCC GCG CCT TCG CTC CTC TAG TGA GGG GAA TTC ACT GAT GAC ACT ATT ACT
-3', anti-sense, 5'- CTA GAG TAA TAG TGT CAT CAG TGA ATT CCC CTC ACT AGA
GGA GCG AAG GCG CGG CCG CTA GTT T -3') was annealed and cloned into the same
vector as above.
cDNA fragments encoding Marf1 (NM_001081154.2; 1838-2646, Primers: forward, 5'- ATT
CTC CGC TTC ATA AAC CA-3', reverse, 5'- ACC TGG CCA TGC TTG GAA AA-3'), Nde1
(NM_023317.2; 313-1513, Primers: forward, 5'- AGG AAC CGG GAC CTC TTG TC -3',
reverse, 5'- GAC AGC TTC AGC TGG AAG CA -3'), Fopnl (NM_025345.2; 1-878, Primers:
forward, 5'-GCC GTT GAA AAA TGG CGA CTG TTA CG, reverse, 5'- GAC AGC TTC
AGC TGG AAG CA), Abcc1 (NM_010275.2; 4800-5799, Primers: forward, 5'-ATC TGA CCT
TGA AGA AGC TG-3', reverse, 5'- AAT AAA TAC ATA TAT TGC CAC C-3'), and Myh11
(NM_013607.2; 5565-6585, Primers: forward, 5'- GGT GCT GTC AAA GCC AAG CT-3',
reverse, 5'-ATT AGT GAA AGT GAC CAT GGG TGC-3') were obtained by polymerase chain
reaction (PCR) from the cDNA of cortical neurons from E14.5 mice cortices. The amplified
products were subcloned into pGEM-T easy vectors (Promega). These vectors were used for
production of riboprobes after restriction enzyme digestion.