96 well plateでのHigh-throughput Screeningui-tei.rnai.jp/assets/files/pdf/RNAiScreeningData061115...0 5 10 15 20 25 30 0 50 100 150 200 250 300 350 400 NAB.4-R1 NAB.4-R2 NAB.4-R3

96 well plateでのHigh-throughput Screening

RNAi効果に影響を与える遺伝子群のスクリーニング

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<Cell density> 1 x 105 cells/ml → 170 µl/well <Transfection> Opti-MEM 10 µl pGL3-Cont 150 nl pRL-SV40 15 ng shRNA + Opti-MEM 10 µl FL2000 0.4 µl Medium Change 50 µl <Assay> PLB=30 µl ３回の実験で、誤差を確認。 (HeLa)

GN.NAB.4

0

5

10

15

20

25

30

0 50 100 150 200 250 300 350 400

NAB.4-R1

NAB.4-R2

NAB.4-R3

binding.07

NAB.3

NTB.7

TF.4

TF.5

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0

10

20

30

40

50

0 50 100 150 200 250 300 350 400

U575

U588

U753

H95

H129

Distribution of Renilla luciferase activity (each experiment)

Renilla luciferase activity (%)

Sam

ple

No.

Sam

ple

No.

Sam

ple

No.

Distribution of Renilla luciferase activity (total)

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Sam

ple

No.

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Sam

ple

No. Luc Assay (379)-10

50 150

GN.NAB.4

0

20

40

60

80

100

120

ABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGH 1 2 3 4 5 6 7 8 9 10 11 12

Rela

tive

luc

activ

ity (%

)

Control pSUPER-FL774 pSUPERretroPuro

EIF2C2

1000

10000

100000Renilla

luc

activ

ity

EIF3S6IP

EIF5

ANKHD1

HBS1L

DBF4

BOLL

CPEB1

ZNF385 EEFSEC

BRF2 UPF2 EIF2C1 SYNJ2

060908-15

0

20

40

60

80

100

120

1000

10000

100000


ABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCD

1 2 3 4 5 6 7 8 9 10 11 12

GN.NAB.3 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity

PUM2

EXOSC8 POLR2G

RBM3 SFRS14

RBM15 �

METTL3

MGC4562

MOV10L1

MSI1

NOL5A

NSUN6

NUFIP1

NXF1

NXF3 OAS2

OAS3

SF3B14

PABPC5

PAPOLA

PNPT1

PSMA1 PUM1

RBMS3 RDBP

RNASE1 RNASEH2A

RNASET2 RPL4

RPN1

RPS21

RPS4Y2 SF3A2

SFRS3 SKIV2L

SNRPA1

060926

0

20

40

60

80

100

120

��

��

��



1 2 3 4 5 6 7 8 9 10 11 12

GN.NTB.7 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity

ROCK1

PRKG1

PSKH2

PSMC1

PSMC6

PTK2

PTK6

PTK7 RET

ARHGAP17

ROCK2

ROS1

RPS6KA1

RPS6KB2 SGK2

CLPB SMC1A SMC5 SPATA5

PRKCD

PRKCG

PRKCH

PKN1

PKN2

PRKD3

PRKCQ

WNK1

WNK2

WNK4 ATAD2

RAD50 RARSL

RPS6KA6

SNF1LK SRMS

SRPK2 STK10

STK16

STK17A

STK19 TSSK3

060929

PRKG2

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1 2 3 4 5 6 7 8 9 10 11 12 � � � �

��

��

GN.TF.4 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity

Rb1 NR2E1

NR2F1

PKNOX1

PPARG

RAX

SATB1

NR2F2

NR5A1

GATAD2A

GATAD2B

PBX1

PBX2 PGR

JARID1A REL

RERE

POU6F2 RUNX1 RUNX2

SCMH1

SCML2

060929

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��

��

��

��

��

��

��

��

��


ABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHEFGH

1 2 3 4 5 6 7 8 9 10 11 12

GN.TF.5 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity

p53

SOX6

SRF

STAT4

TBX6

TCEA1 TBX4

060929

SLC38A6

SNAPC5

SOX2

SOX5

SPIC

SUPT6H

T

TADA3L TAF13 TAF5L TBX1

TBX5 TCF3

MLX

TEAD4

TFAP2C

THRA

TNRC4

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��

��

��

��

��

��

1000

10000

100000

Rbbp6

GN.binding.07 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity



1 2 3 4 5 6 7 8 9 10 11 12

LRP6

LRPAP1 SMAD7 NMNAT1

PGLYRP3

KBTBD10

SLC9A3R2 SGTA SENP2 SOCS5

SORCS2

CDC42SE1 SPOP

PEA15

GIPC1

LMNA LTBP4

RPE65 MPP6

RTKN

060926

0

20

40

60

80

100

120

1000

10000

100000

GN.binding.07-2 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control

pSUPER-retro-puro EIF2C2


1 2 3 4 5 6 7 8 9 10 11 12

Rbbp6 LRP6 LRPAP1

SMAD7 MPP2 NMNAT1

PGLYRP3

SLC9A3R2 SOCS5

SORCS2 SPOP

MPP6

NPHP1

NUDT5

OIP5 PHIP RCBTB1

RCN2

GIPC1

RIN1

SENP2

RPE65

SLIT3 SMO

SNCAIP

CDC42SE1

TCEB3

061013

0

20

40

60

80

100

120GN.binding.07-1GN.binding.07-2

1000

10000

100000

!"#$%&'%&(#)*+,

!"#$%&'%&(#)*+-

Rbbp6

GN.binding.07-1&2 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity



1 2 3 4 5 6 7 8 9 10 11 12

LRP6

LRPAP1 SMAD7 NMNAT1

PGLYRP3

KBTBD10

SLC9A3R2 SGTA SENP2 SOCS5

SORCS2

CDC42SE1 SPOP

PEA15

GIPC1

LMNA LTBP4

RPE65 MPP6

RTKN

0

20

40

60

80

100

120

1000

10000

100000

GN.NTB.2 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

CAMK1G CASK

CDK2

CDK4

CDKL2

CDKL5 YARS2 CLK3

DDR2 BMP2K

TRIB1 CACNA1B

BMPR1B

BMPR2

BUB1 NUBPL

TSSK4

CAMK1

CAMK2A CCT7

CDC7 CLK1 CLK4

CSNK1E

CSNK1G3

061013

CDC2

0

20

40

60

80

100

120

1000

10000

100000

GN.DNAB.1 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

SUV39H1

POLH POLQ

CHAF1A

RBMS1 IFI16

SAFB

JTV1

ERCC1 MSH3

MSH5

NBN

RAD1

RAD18 REV1L

XPA XRCC2

CENPA

ING2 CBX2

NCOA6

ARID4B MLH3

061013

0

20

40

60

80

100

120

1000

10000

100000

GN.NAB.2 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control


ABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGH

1 2 3 4 5 6 7 8 9 10 11 12

A2BP1

ADAT1 CSTF3

CUGBP2

HNRPA2B1

KIAA0427

LGTN

DDX21 DDX43

EIF2S1 CIRBP

BAT1

061017

EIF4G1

DAZL EIF4G3

EIF4E2

0

20

40

60

80

100

120

STAU1

1000

10000

100000

GN.NAB.1 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control


ABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCDEFGHABCD 1 2 3 4 5 6 7 8 9 10 11 12

WDR57 KHSRP

LSM8 LSMD1

SF3B1

SNRPB

SFRS11

SNRPN

EFTUD2

ELAVL4

ADARB2

CARF

DICER1

LRRFIP1

PRKRA TARBP2

SF3B4 LSM11 AUH

CPSF1 ELAVL2

RNASEN (DROSHA)

DUS2L

061017

PABPC3

PABPC4

STAU2

ADAD1

0

20

40

60

80

100

120

1000

10000

100000

GN.Hs_71_TF.1 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

AHR

ALX4

AR ARNTL

ATF7

ATOH1 BNC1

BRD8

BRF1

BTAF1

NULL

TSHZ2 RUNX1T1

CEBPB

ARNT ARNT2

ARNTL2

ASCL1 ATF3

ATF6 ATRX BACH1

BAPX1

BARX1

BARX2

MKX

CART1

CBFA2T3

CBFB

061017

CDX2

CDX4

0

20

40

60

80

100

120

1000

10000

100000

GN.A_L.08 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

ALKBH3

PSRC1 DC-UbP

PCBD2

DCDC2

ENY2

C3orf37

DIDO1

CD55

CYP26C1

CYFIP1

CYBRD1

CXorf6 CTLA4 SLC44A2

061027

DCP1B DCPS

DCP2

0

20

40

60

80

100

120

1000

10000

100000

GN.NAB.5 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

DDX42

DDX51

DDX54 DHX35

ADPGK

ZNF540 DDX59

DRB1

GPATC1

ENDOGL1

ENPP2

ZNF669 ZNF649

RBM35A

RBM21

ZNF419

NULL ZNF582

C3orf33 ZNF641

ZNF548

ZNF597

DDX19A

061027

0

20

40

60

80

100

120

1000

10000

100000

GN.NAB.6 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

DHX33

DHX40

KIAA1429

EHD2

WDR52

ZC3H14

ZNF669 ZNF649

ZNF566

FLJ20035

ZNF586 RBED1 ZNF671

061027

IFIH1

0

20

40

60

80

100

120

1000

10000

100000

GN.NAB.7 Re

lativ

e lu

c ac

tivity

(%)

Renilla

luc

activ

ity Control



1 2 3 4 5 6 7 8 9 10 11 12

061110

RAVER1

DDX58 RNASE2 RBM24

RSN

SART3

TRSPAP1

SFRS12

SPEN THOC4

TIPARP U1SNRNPBP

USP6

PARP12

ZCCHC2

RNF113B

ZNF227

ZNF354C

ZNF415

ZNF426

ZNF454

ZNF471

0

20

40

60

80

100

120

GN

.NA

B.4

-1 (H

9)

GN

.NA

B.4

-2 (H

9)

GN

.NA

B.4

-3 (H

9)

GN

.bin

din

g.0

7 (E

12

)G

N.N

AB

.3 (E

12

)G

N.N

TB

.7 (E

12

)G

N.T

F.4 (E

12

)G

N.T

F.5 (A

12

)G

N.b

ind

ing

.07

-2 (A

1)

GN

.bin

din

g.0

7-2

(C5

)G

N.b

ind

ing

.07

-2 (C

9)

GN

.bin

din

g.0

7-2

(E1

2)

GN

.NT

B.2

(A1

)G

N.N

TB

.2 (C

5)

GN

.NT

B.2

(C9

)G

N.N

TB

.2 (E

12

)G

N.D

NA

B.1

(A1

)G

N.D

NA

B.1

(C5

)G

N.D

NA

B.1

(C9

)G

N.D

NA

B.1

(E1

2)

GN

.NA

B.2

(E1

)G

N.N

AB

.2 (C

4)

GN

.NA

B.2

(D8

)G

N.N

AB

.2 (A

12

) G

N.N

AB

.1 (A

1)

GN

.NA

B.1

(C5

)G

N.N

AB

.1 (C

9)

GN

.NA

B.1

(E1

2)

GN

.Hs_

71

_T

F.1 (A

1)

GN

.Hs_

71

_T

F.1 (C

4)

GN

.A_

L.0

8 (A

1)

GN

.A_

L.0

8 (C

5)

GN

.A_

L.0

8 (C

9)

GN

.A_

L.0

8 (E

12

)G

N.N

AB

.5 (C

3)

GN

.NA

B.5

(C6

)G

N.N

AB

.5 (C

9)

GN

.NA

B.5

(E1

2)

GN

.NA

B.6

(A1

)G

N.N

AB

.6 (C

5)

GN

.NA

B.6

(B9

)G

N.N

AB

.6 (E

12

)

GN

.NA

B.4

-1 (H

10

)G

N.N

AB

.4 -2

(H1

0)

GN

.NA

B.4

-3 (H

10

)G

N.b

ind

ing

.07

(F1

2)

GN

.NA

B.3

(F1

2)

GN

.NT

B.7

(F1

2)

GN

.TF.4

(F1

2)

GN

.TF.5

(B1

2)

GN

.NA

B.4

-1 (H

11)

GN

.NA

B.4

-2 (H

11)

GN

.NA

B.4

-3 (H

11)

GN

.bin

din

g.0

7 (G

12

)G

N.N

AB

.3 (G

12

)G

N.N

TB

.7 (G

12

)G

N.T

F.4 (G

12

)G

N.T

F.5 (C

12

)G

N.b

ind

ing

.07

-2 (B

1)

GN

.bin

din

g.0

7-2

(D5

)G

N.b

ind

ing

.07

-2 (D

9)

GN

.bin

din

g.0

7-2

(F1

2)

GN

.NT

B.2

(B1

) G

N.N

TB

.2 (D

5)

GN

.NT

B.2

(D9

)G

N.N

TB

.2 (F

12

)G

N.D

NA

B.1

(B1

) G

N.D

NA

B.1

(D5

)G

N.D

NA

B.1

(D9

)G

N.D

NA

B.1

(F1

2)

GN

.NA

B.2

(F1

)G

N.N

AB

.2 (D

4)

GN

.NA

B.2

(E8

)G

N.N

AB

.2 (B

12

)G

N.N

AB

.1 (B

1)

GN

.NA

B.1

(D5

)G

N.N

AB

.1 (D

9)

GN

.NA

B.1

(F1

2)

GN

.Hs_

71

_T

F.1 (B

1)

GN

.Hs_

71

_T

F.1 (D

4)

GN

.A_

L.0

8 (B

1)

GN

.A_

L.0

8 (D

5)

GN

.A_

L.0

8 (D

9)

GN

.A_

L.0

8 (F

12

)G

N.N

AB

.5 (D

3)

GN

.NA

B.5

(D6

)G

N.N

AB

.5 (D

9)

GN

.NA

B.5

(F1

2)

GN

.NA

B.6

(B1

)G

N.N

AB

.6 (D

5)

GN

.NA

B.6

(C9

)G

N.N

AB

.6 (F

12

)

GN

.bin

din

g.0

7-2

(C1

)G

N.b

ind

ing

.07

-2 (E

5)

GN

.bin

din

g.0

7-2

(E9

)G

N.b

ind

ing

.07

-2G

N.N

AB

.2 (C

1)

GN

.NA

B.2

(E5

)G

N.N

AB

.2 (E

9)

GN

.NA

B.2

(G1

2)

GN

.DN

AB

.1 (C

1)

GN

.DN

AB

.1 (E

5)

GN

.DN

AB

.1 (E

9)

GN

.DN

AB

.1 (G

12

)G

N. N

AB

.2 (G

1)

GN

. NA

B.2

(E4

)G

N. N

AB

.2 (F

8)

GN

. NA

B.2

(C1

2)

GN

. NA

B.1

(C1

)G

N. N

AB

.1 (E

5)

GN

. NA

B.1

(E9

)G

N. N

AB

.1 (G

12

)G

N.H

s_7

1_

TF.1

(C1

)G

N.H

s_7

1_

TF.1

(E4

)G

N.A

_L

.08

(C1

) G

N.A

_L

.08

(E5

)G

N.A

_L

.08

(E9

)G

N.A

_L

.08

(G1

2)

GN

.NA

B.5

(E3

)G

N.N

AB

.5 (E

6)

GN

.NA

B.5

(E9

)G

N.N

AB

.5 (G

12

)G

N.N

AB

.6 (C

1)

GN

.NA

B.6

(E5

)G

N.N

AB

.6 (D

9)

GN

.NA

B.6

(G1

2)

control Re

lativ

e lu

c ac

tivity

(%)

Luc. Luc. + pSUPER -FL774

Luc. + pSUPER-FL774 + pSUPER-retro-puro

Luc. + pSUPER-FL774 + EIF2C2

0

20

40

60

80

100

120

GN

.bin

din

g.0

7-2

(A1

)G

N.N

TB

.2 (A

1)

GN

.DN

AB

.1 (A

1)

GN

.NA

B.1

(A1

)G

N.H

s_7

1_

TF.1

(A1

)G

N.A

_L

.08

(A1

) G

N.N

AB

.6 (A

1)

GN

.TF.5

(A1

2)

GN

.NA

B.2

(A1

2)

GN

.NA

B.6

(B9

)G

N.N

AB

.5 (C

3)

GN

.NA

B.2

(C4

)G

N.H

s_7

1_

TF.1

(C4

)G

N.b

ind

ing

.07

-2 (C

5)

GN

.NT

B.2

(C5

)G

N.D

NA

B.1

(C5

)G

N.N

AB

.1 (C

5)

GN

.A_

L.0

8 (C

5)

GN

.NA

B.6

(C5

)G

N.N

AB

.5 (C

6)

GN

.bin

din

g.0

7-2

(C9

)G

N.N

TB

.2 (C

9)

GN

.DN

AB

.1 (C

9)

GN

.NA

B.1

(C9

)G

N.A

_L

.08

(C9

)G

N.N

AB

.5 (C

9)

GN

.NA

B.2

(D8

)G

N.N

AB

.2 (E

1)

GN

.bin

din

g.0

7 (E

12

)G

N.N

AB

.3 (E

12

)G

N.N

TB

.7 (E

12

)G

N.T

F.4 (E

12

)G

N.b

ind

ing

.07

-2 (E

12

)G

N.N

TB

.2 (E

12

)G

N.D

NA

B.1

(E1

2)

GN

.NA

B.1

(E1

2)

GN

.A_

L.0

8 (E

12

)G

N.N

AB

.5 (E

12

)G

N.N

AB

.6 (E

12

)G

N.N

AB

.4 -1

(H9

)G

N.N

AB

.4 -2

(H9

)G

N.N

AB

.4 -3

(H9

)

GN

.TF.5

(B1

2)

GN

.bin

din

g.0

7 (F

12

)G

N.N

AB

.3 (F

12

)G

N.N

TB

.7 (F

12

)G

N.T

F.4 (F

12

)G

N.N

AB

.4 -1

(H1

0)

GN

.NA

B.4

-2 (H

10

)G

N.N

AB

.4 -3

(H1

0)

GN

.bin

din

g.0

7-2

(B1

)G

N.N

TB

.2 (B

1)

GN

.DN

AB

.1 (B

1)

GN

.NA

B.1

(B1

)G

N.H

s_7

1_

TF.1

(B1

)G

N.A

_L

.08

(B1

)G

N.N

AB

.6 (B

1)

GN

.NA

B.6

(C9

)G

N.T

F.5 (C

12

)G

N.N

AB

.5 (D

3)

GN

.NA

B.2

(D4

)G

N.H

s_7

1_

TF.1

(D4

)G

N.b

ind

ing

.07

-2 (D

5)

GN

.NT

B.2

(D5

)G

N.D

NA

B.1

(D5

)G

N.N

AB

.1 (D

5)

GN

.A_

L.0

8 (D

5)

GN

.NA

B.6

(D5

)G

N.N

AB

.5 (D

6)

GN

.bin

din

g.0

7-2

(D9

)G

N.N

TB

.2 (D

9)

GN

.DN

AB

.1 (D

9)

GN

.NA

B.1

(D9

)G

N.A

_L

.08

(D9

)G

N.N

AB

.5 (D

9)

GN

.NA

B.6

(C9

)G

N.N

AB

.2 (E

8)

GN

.NA

B.2

(F1

)G

N.b

ind

ing

.07

-2 (F

12

)G

N.N

TB

.2 (F

12

)G

N.D

NA

B.1

(F1

2)

GN

.NA

B.1

(F1

2)

GN

.A_

L.0

8 (F

12

)G

N.N

AB

.5 (F

12

)G

N.N

AB

.6 (F

12

)G

N.b

ind

ing

.07

(G1

2)

GN

.NA

B.3

(G1

2)

GN

.NT

B.7

(G1

2)

GN

.TF.4

(G1

2)

GN

.NA

B.4

-1 (H

11)

GN

.NA

B.4

-2 (H

11)

GN

.NA

B.4

-3 (H

11)

GN

.bin

din

g.0

7-2

(C1

)G

N.N

AB

.2 (C

1)

GN

.DN

AB

.1 (C

1)

GN

. NA

B.1

(C1

)G

N.H

s_7

1_

TF.1

(C1

)G

N.A

_L

.08

(C1

) G

N.N

AB

.6 (C

1)

GN

. NA

B.2

(C1

2)

GN

.NA

B.6

(D9

)G

N.N

AB

.5 (E

3)

GN

. NA

B.2

(E4

)G

N.H

s_7

1_

TF.1

(E4

)G

N.b

ind

ing

.07

-2 (E

5)

GN

.NA

B.2

(E5

)G

N.D

NA

B.1

(E5

)G

N. N

AB

.1 (E

5)

GN

.A_

L.0

8 (E

5)

GN

.NA

B.6

(E5

)G

N.N

AB

.5 (E

6)

GN

.bin

din

g.0

7-2

(E9

)G

N.N

AB

.2 (E

9)

GN

.DN

AB

.1 (E

9)

GN

. NA

B.1

(E9

)G

N.A

_L

.08

(E9

)G

N.N

AB

.5 (E

9)

GN

. NA

B.2

(F8

) G

N. N

AB

.2 (G

1)

GN

.bin

din

g.0

7-2

GN

.NA

B.2

(G1

2)

GN

.DN

AB

.1 (G

12

)G

N. N

AB

.1 (G

12

)G

N.A

_L

.08

(G1

2)

GN

.NA

B.5

(G1

2)

GN

.NA

B.6

(G1

2)

Rela

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Luc. Luc. + pSUPER -FL774

Luc. + pSUPER-FL774 + pSUPER-retro-puro

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Control.2 (position)

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1 2 3 4 5 6 7 8 9 10 11 12

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INTS6

PAPD1

ZNF549

ZNF554

STK31

Documents

96 well plateでのHigh-throughput Screeningui-tei.rnai.jp/assets/files/pdf/RNAiScreeningData061115...0 5 10 15 20 25 30 0 50 100 150 200 250 300 350 400 NAB.4-R1 NAB.4-R2 NAB.4-R3