supplementary Fig1.1 for MP - media.nature.com · ncp mir-484p ncp mir-484p nci mir-484i nci mir-484i 1 div 2 div c ... cta gaa ggc ttc cct tca ggc tcc tgg gcc ctg agc gct ctt gct

Supplementary Figure 1Establishment of the gain- and loss-of-function experiments and cell survival assays.

0.0

0.5

1.0

1.5

2.0

2.5

0

10

20

30

ba

1 DIV 2 DIV

Rel

ativ

e ex

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atur

e m

iR-4

84

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atur

e m

iR-4

84

******

**

**** ****

NCP miR-484P NCP

miR-484P NCI

miR-484I NCI

miR-484I

1 DIV 2 DIV

cTuj1(-) Tuj1(+)

apoptotic live

Tuj1

/GF

P/D

AP

ITu

j1G

FP

DA

PI

Tuj1

/GF

P/D

AP

ITu

j1G

FP

DA

PI

apoptotic live

MOCKPCDH19-myc

+ - - +

+ - - +

WB:anti-mycAntibody

225

1501027652

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225

1501027652

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12

MOCKPCDH19-myc

scr sh10.0

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PCD

H19

mR

NA

***b

a

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.

15

1515

15

15

15

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.