c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP
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1 Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir **** **** NCP mir- 484P NCP mir- 484P b Relative expression of mature mir NCI ** ** mir- 484I NCI **** mir- 484I 1 DIV 2 DIV 1 DIV 2 DIV c Tuj1(-) apoptotic live apoptotic Tuj1(+) live Tuj1/GFP/DAPI Tuj1 DAPI GFP Tuj1/GFP/DAPI Tuj1 DAPI GFP
2 Supplementary Figure 2 Assessment of the PCDH19 antibody and Pcdh19 shrna vector. a WB: anti-myc Antibody WB: anti-pcdh19 Antibody MOCK PCDH19-myc MOCK PCDH19-myc b 1.5 *** Relative expression of PCDH19 mrna scr sh1
3 Supplementary Table 1 Clinical manifestation data obtained from the DECIPHER/ISCA databases and reported papers.
4 Supplementary Materials and Methods Plasmids Full-length cdna fragments encoding mouse Nde1 (NM_ ; , 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_ ; 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_ , 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
5 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
6 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
7 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
8 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.
9 cdna fragments encoding Marf1 (NM_ ; , Primers: forward, 5'- ATT CTC CGC TTC ATA AAC CA-3', reverse, 5'- ACC TGG CCA TGC TTG GAA AA-3'), Nde1 (NM_ ; , Primers: forward, 5'- AGG AAC CGG GAC CTC TTG TC -3', reverse, 5'- GAC AGC TTC AGC TGG AAG CA -3'), Fopnl (NM_ ; 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_ ; , 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_ ; , 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.
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