1 PI: Dr. James C.-K. Shen ( 沈哲鯤 ) Co-PI: Dr. Michael Hsiao ( 蕭宏昇副研究員 ) Co-PI/ Manager: Dr. King-Song Jeng ( 鄭金松副技師 ) Speaker: King-Song Jeng ( 鄭金松

1

PI: Dr. James C.-K. Shen ( 沈哲鯤 )Co-PI: Dr. Michael Hsiao ( 蕭宏昇副研究員 )

Co-PI/ Manager: Dr. King-Song Jeng ( 鄭金松副技師 )

Speaker: King-Song Jeng ( 鄭金松 )

Institute of Molecular Biology / Genomic Research Center Academia Sinica

Sponsored by National Research Program for Genomic Medicine, National Science Council / Academia Sinica

C6-The RNAi Core 10/16/98

2

Outline

• The RNAi Consortium (TRC)

• Current status of the RNAi Core

• Collaboration Research Project

3

The RNAi Consortium (TRC)

Objectives: Create genome wide, renewable RNAi reagents for research and educational uses;

Develop, validate and optimize materials, methodologies and information for their effective application in research.

4

The RNAi Consortium (TRC) The RNAi Core

Phase I (May/2004 to Apr/2007) Jun/2005 to Apr/2008

Phase II (Oct/2007 to Sept/2011) May/2008 to Apr/2011

Phases of TRC Program

5

Sponsoring Members Collaborating Laboratories

Taiwan, ROC +Companies in USA:TRC-I:– Bristol-Myers Squibb– Eli Lilly– Novartis– Sigma – Aldrich

TRC-II:– Broad Institute of MIT/

Harvard– Ontario Institute for Cancer

Research – Bristol-Myers Squibb– Sigma – Aldrich

1. Nir Hacohen, Whitehead Institute, HMS,

Massachusetts General Hospital

2. David Sabatini, Whitehead Institute/MIT

3. Stuart Schreiber, Harvard University, Broad Institute

4. Sheila Stewart, Washington University

5. Brent Stockwell, Whitehead Institute 6. David Bartel, Whitehead Institute/MIT

7. Todd Golub, Dana-Farber Cancer

Institute, HMS*, Broad Institute

8. Bill Hahn, Dana-Farber Cancer Institute, HMS

9. Ed Harlow/Josh LaBaer, Harvard Institute for Proteomics, HMS

10. Eric Lander, Broad Institute, Whitehead/MIT/Harvard

Composition of TRC-I and TRC-II

* HMS: Harvard Medical School

6

Lentivirus-based RNAi(VSV-G peudotyped virus)

http://www.accessexcellence.org/RC/VL/GG/retrovirus.html

shRNA

7

Vector Used by TRC/RNAi Core

http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Functional_Genomics_and_RNAi/Product_Lines/shRNA_Library.html

EcoRI

(GAATTC)

AgeI

(ACCGGT)

TRC2 library goals

More powerful library: – enriched for best OT KD shRNAs– even coverage of genome

TRC2 library3+ good shRNAs per gene

High-performing existing clones

New clones(test)

9

Materials Received from TRC

shRNA constructs and knockdown information:

TRC-I TRC-II

Clone # Gene # KD Clone # Gene # KD

Human 81,888 16,026 34,893 46,428 5,516 5,040

Mouse 77,700 15,976 32,188 31,202 4,389 1,753

Control 85 129

Total 159,588 32,002 67,081 77,759 9,905 6,793

Pooled genome-wide shRNA plasmid DNAs and chips (human and mouse) for RNAi genome-wide screening.

10

67,000 TRC shRNAs targeting 12,200 genes(Jurkat data excluded)

J. Grenier

56%

73%

02000400060008000

10000

1200014000160001800020000

<=10% <=20 <=30% <=50% No KD

Clone Performance (% expression)

#sh

RN

A

0%

20%

40%

60%

80%

100%

120%

Cu

mu

lati

ve %

sh

RN

A

62%

76%88%

0

5001000

1500

20002500

3000

35004000

4500

all HPS ~4/5 ~3/5 ~2/5 ~1/5 ~0/5

% "good" clones/gene#

gen

es

0%

20%

40%

60%

80%

100%

120%

Cu

mu

lati

ve %

gen

es

All Hairpins Statistics Per Gene Statistics

TRC shRNA performance stats: Sept.09

How reproducible are the data?:How consistent is the > 70% call?

Trial 1

Tria

l 2

>70%KD?

+ –

+1358(39%)

498(14%)

–391

(11%)1248(36%)

Total hairpins: 3495

Jen Grenier, Shuba Gopal

Consistent ‘pass call’ 75% of the time.

33% of inconsistent calls are > 60% KD in the failed rep

50% of inconsistent calls are < 80% KD in the passed rep

12

TRC TAIWANRNAi

CORE

13

National RNAi Core (Since June 2005)

Housed in Genomics Research Center (GRC) and Institute of Molecular Biology (IMB)

Connected to TRC-I and TRC-II Responsible for Services Technology R&D Collaborative research activities

Welcome local and international collaborations

14

Aims of RNAi Core: Third Phase

Objectives:

Maximizing the utilization of the RNAi library in mammalian genetic screen

15

Routine Service Items

Categories Service Items

Bacterial Clones shRNA construct

VSV-G lentivirus

Arrayed VSV-G pseudotyped lentivirus

Pooled VSV-G pseudotyped lentivirus

Individual VSV-G pseudotyped lentivirus

Customized lentivirus HT&HCS Image Analysis

HT&HCS Image Analysis

Plasmid DNA(lentiviral transfer vector)

Package plasmids

shRNA cloning lentivector

Pol II/ gene expression lentivector*

16

Total Users: 266 PIs (Feb 2008 to Jan 2009)Total Users: 266 PIs (Feb 2008 to Jan 2009)

Bacterium: 71%Bacterium: 71%Virus: 8%Virus: 8%HTC image analysis: 3%HTC image analysis: 3%LentiviralLentiviral vector: 19%vector: 19%

2%2%

71%71%

8%8% 19%19%

Analyses of Geographic Distribution and Distributed Items to the Users

Total Users: 465 PIs

Bacterium: 79%Virus: 6%

HTC image analysis: 2%

Lentiviral vector: 13%

2%

79%

6% 13%

Total Users: 465 PIs

11%

58%

3%20%

8%

Academia Sinica(AS• ): 11%N. Taiwan(AS not included): 58%Middle Taiwan: 8%Southern Taiwan: 20%Eastern Taiwan: 3%

17

User Publication

25

54

0246

8101214161820

<5 5~10 >10

Impact Factor#

of

pu

bli

cat

ion

6

16

0

2

46

8

10

12

1416

18

20

2006~2007 2008~2009Year

# o

f p

ub

lic

atio

n

12

2009~2010

Please acknowledge the RNAi Core for RNAi reagents whenyour research is get published.

Please refer to http://rnai.genmed.sinica.edu.tw/faq-detail.asp?sn=16for example.

http://rnai.genmed.sinica.edu.tw/faq-detail.asp?sn=16

18

Items Plan to Be Served in the Future

Genome-wide RNAi pooled screening

Produce/ provide shRNA-expressing lentivirus defined by User (a 2D barcode shRNA plasmid DNAs library is being established)

Enlarge the activity of RNAi library screening

• C5: perform microarray and data analysis• C6: provide pooled virus

19

Collaborative Research Project(s)workflow and regulations

Current regulations: Tying with the workload of the Core ;

Sharing cost and labor;

Yes

No Reject or Revise

Yes

Submission Re-submission

RNAi screen and data analysis

Data arrangementand release

Discuss on thePotential project

Review bycommittee

Case closed

Notifyuser committee

Future capacity Could accommodate more projects if transform Core into National RNAi Screening Center (NRSC).

20

Contact Information

中央研究院基因體研究中心四樓 RNAi Core Lab.

核心電話 : 02-27899724 ( 分機 15) 鍾穎麗小姐核心傳真 : 02-27885420

服務信箱 : [email protected]

核心網址 : http://rnai.genmed.sinica.edu.tw/index.asp

核心地址 : 台北市南港區 115 研究院路二段 128 號

mailto:[email protected]

21

Chi-long Lin

Navigation of RNAi Website

Navigation of RNAi website

–Overview–Search function–Knockdown information

database–New Online Ordering system–Feedback mechanism




cloneId TRC KnockDown symbol nmId NCBI_geneId

TRCN0000019199 COPS5 NM_006837 10987

TRCN0000019200 COPS5 NM_006837 10987

TRCN0000019201 COPS5 NM_006837 10987

TRCN0000019202 COPS5 NM_006837 10987

TRCN0000019203 COPS5 NM_006837 10987

TRCN0000072558 99% COPS6 NM_006833 10980

TRCN0000072559 79% COPS6 NM_006833 10980

TRCN0000072560 91% COPS6 NM_006833 10980

TRCN0000072561 99% COPS6 NM_006833 10980

TRCN0000072562 99% COPS6 NM_006833 10980

TRCN0000073983 97% EIF3F NM_003754 8665

TRCN0000073984 85% EIF3F NM_003754 8665

TRCN0000073985 88% EIF3F NM_003754 8665

TRCN0000073986 98% EIF3F NM_003754 8665

TRCN0000073987 99% EIF3F NM_003754 8665

TRCN0000073988 EIF3FP2 NM_031943 83880

TRCN0000073989 EIF3FP2 NM_031943 83880

TRCN0000073990 EIF3FP2 NM_031943 83880

TRCN0000073991 EIF3FP2 NM_031943 83880

TRCN0000073992 EIF3FP2 NM_031943 83880

TRCN0000073978 96% EIF3H NM_003756 8667

TRCN0000073979 35% EIF3H NM_003756 8667

TRCN0000073980 95% EIF3H NM_003756 8667

TRCN0000073981 95% EIF3H NM_003756 8667

TRCN0000073982 80% EIF3H NM_003756 8667

TRCN0000004427 70% PAN2 NM_014871 9924

TRCN0000004428 74% PAN2 NM_014871 9924

TRCN0000004429 84% PAN2 NM_014871 9924

TRCN0000004430 72% PAN2 NM_014871 9924

TRCN0000010875 84% PAN2 NM_014871 9924

TRCN0000004535 PARP11 NM_020367 57097

TRCN0000004536 PARP11 NM_020367 57097

TRCN0000004537 PARP11 NM_020367 57097

TRCN0000004538 PARP11 NM_020367 57097

TRCN0000010887 PARP11 NM_020367 57097

TRCN0000075108 97% PRPF8 NM_006445 10594

TRCN0000075109 94% PRPF8 NM_006445 10594

TRCN0000075110 PRPF8 NM_006445 10594

TRCN0000075111 91% PRPF8 NM_006445 10594

TRCN0000075112 99% PRPF8 NM_006445 10594

TRCN0000006455 42% PSMD14 NM_005805 10213

TRCN0000006456 71% PSMD14 NM_005805 10213

TRCN0000006457 69% PSMD14 NM_005805 10213

TRCN0000011023 59% PSMD14 NM_005805 10213

TRCN0000058063 100% PSMD7 NM_002811 5713

TRCN0000058064 97% PSMD7 NM_002811 5713

TRCN0000058065 44% PSMD7 NM_002811 5713

TRCN0000058066 -59% PSMD7 NM_002811 5713

TRCN0000073963 STAMBPL1 NM_020799 57559

TRCN0000073964 STAMBPL1 NM_020799 57559




TRC validation progress: Sept.09

0

5,000

10,000

15,000

20,000

25,000

30,000

Septe

mber

Febru

ary

April

May

June

July

August

Septe

mber

Octo

ber

Novem

ber

Decem

ber

January

Febru

ary

Marc

h

April

May

June

July

August

Septe

mber

Octo

ber

Novem

ber

Decem

ber

January

Febru

ary

Marc

h

April

May

June

July

August

2006 2007 2008 2009

# g

en

es (

cu

mu

lati

ve)

successful

attempted

J. Grenier, X. Yang

Recent Pace:- Attempts: 1,000 genes/month- Successes: 750 genes/month

Cumulative:67,000 TRC shRNAs Targeting 12,200 genes







Issues Regarding Lentiviral Transfer Vectors and shRNA/siRNA-

triggered Off-Target

Speaker: 鄭金松 , RNAi Core Manager

98/10/16

3’LTRpLKO_AS2

EMCVIRES25’LTR puro

Maps of lentivirus-based cDNA- expressing transfer vectors

CMVie p

CAG p

Ubiqui p

hPGK p

EF1a p

eGFP

pLKO_AS3w

pLKO_AS6w

pLKO_AS7w

pLEX_TRC203

0.6 kb

1.7 kb

1.2 kb

0.5 kb

1.26 kb

PCR Amplification of DNA Fragments Sticky-End PCR Method

PCR5’-CTAGC3’-GATCG

5’-C3’-G

-3’-5’

-3’-5’

&

Purify and mix 2 PCR products in an equimolarratio; then de-nature and re-nature

5’-CTAGC -3’3’-G -5’

(Effective annealing PCR product for ligation)

Primer #15’-CTAGC

5’ #Primer #2Primer #1

5’-CTAGC

5’ #Primer #2

Separated PCR tube #1

Separated PCR tube #2

# Both reactions use the same reverse primer for PCR amplification.

0 10 20 30 40 50 606

6.5

7

7.5

8

8.5

9

9.5

AS2 AS3W AS6W AS7W TRC203AS2+P AS3W+P AS6W+P AS7W+P TRC203+P

Days

Arb

itra

ry In

ten

sity

(L

og

)

(CMVp) (CAGp) (Ubqp) (hPGKp) (EF1ap)

Expression Stability of Lentivirus-based System:Effect of Promoters

Cell: A549

Wen-Ya

How long of insert can be tolerated in HIV-1-derived transfer vector?

Genome Length of HIV-1

Potential Insertion Length of pLKO_AS3w.puro Vectors

pLKO AS3w.puro9197 bp

Am p

P AC

HIV 3 '-LTR

HIV 5 '-LTR

O ri

RRE

P s i s eque nce

S V 4 0 P olyA

IRE S 1

Be ta -Actinp

cP P T

WP RE

RS V P romoter

CM V ie E nh

CAG promoter

bla prom oter

A sc I (19 88 )Eco RI (19 95 )

N h e I (19 81 )

P m e I (20 11 )P st I (20 05 )

Sf i I (44 60 )

X h o I (1 )

HIV 5’-LTR-HIV 3’-LTR = 5732bp∼Potential Insertion Size: 9749-5732= 4017bp

Genome Organization of HCV

1kbMarker10k8k6k

3k

M 1 2 3 4 5 6

+ +++ + +++ ++ +++

Insert(kb)

G418-resistantcells

0.8 1.4 1.8 2.1 2.6 3.1

Effects of Insert Length on Virus Production

4B 5A 5B 34A 34AB 5AB

CAG p neo/G418

AS3w.neo

Insert

IRES

1kbMarker

10k

6k

3k

M 1 2 3 4 5 6

Effects of Insert Length on Virus Production

CAG p puro

AS3w.puro

0 1.3 0 3.6 0 2.5

+/- ++ +/- +++ +/- ++

+ ++++ + ++++ + ++++

Insert (kb)

Titer(+E5)

0.8 1.4 1.8 2.1 2.6 3.1

4B 5A 5B 34A 34AB 5ABInsert

IRES

Puro-resistant day 2

Puro-resistant day 5

HA-NS3-

4ANS3-

4AB-H

A

Western Analysis of Lentivirus Transducing Cells Study of the HCV NS Proteins

1 2 3 4 5

Ctl

NS4B-H

A

HA-NS5B

NS5A-H

A

HA-NS3-

4A

Transient Transfection (done by Ti-Chun)

moc

kNS4B

-HA

HA-NS5B

NS5AB-H

A

NS5A-H

A

1 2 3 4 5 6 7

130

957555

43

34

25

kD

Virus Transduction(Done by Huey-Lan)

The data indicate that there is no direct correlation between Virus Titer and Insert Length.

Why?

Is second structure of IRES disrupted by insert sequences?

Summary

Structure of the 5’-UTR of the EMCV IRES

Hum

an PGK p

Replacement of IRES Element by hPGK Promoter

CAG p puro

AS3w.PpurohPGKp

1.9 3.8 4.4 4.6 0 3.7

+2 +3 +3 +3 0 +2.5

++++ ++++ ++++ ++++ + ++++

Insert (kb)

Titer(+E5)

0.8 1.4 1.8 2.1 2.6 3.1

4B 5A 5B 34A 34AB 5ABInsert

Puro-resistant d2

IRES sequence may be disrupted by upstream sequences.

Upstream Insert Sequences Plays a Role in IRES-directed Translation

Puro-resistant d5

Secondary Structure of HIV-1 Psi Sequence

JMB 326: 529, 2008

pLKO.1-puro:

R-U5RSV promoter

Psi signal

RRE CAG promoter

hPGK promoter

Puro

U3-R

SV40 PolyAPPTpbs

SINSIN

cPPT

Cis Elements That Are Required for HIV-1 Replication

R-U5RSV promoter

Psi signal

RRE CAG promoter

hPGK promoter

Puro

U3-R

SV40 PolyAPPTpbs

SINSIN

cPPT

NruI NruI1268bp

Can sequences Be Deleted Without Affecting Virus Replication in AS3w.Ppuro Vector

RRE = 242bpPotential increase of insert length = 1268bp – 242bp = 1026bp

pLAS AS3wd.Ppuro8171 bpAm p

PAC

H IV 3'-LTR

H IV 5'-LTR

Ori

Psi sequence

SV40 Po lyA

B eta-Actin p

W PR E

hPGK

cPPT

RRE

R SV Prom o ter C M VieEnh

C AG p rom oter

bla prom oter

Bsr GI (2020)

Eco RI (2047)

Eco RV (2028)

Nhe I (2014)

Pme I (2035)Sbf I (2045)

Features of New Lentiviral Transfer Vector

HIV 5’-LTR-HIV 3’-LTR = 4706bp∼Potential Insertion Size: 9749-4706= 5043bp

CAG p puro

AS3wd.PpurohPGKp

6.9+E5; 3.6+E5 1.5+E6; 1.9+E6

many many

Insert (kb)

Titer

Insert

Puro resistant cells

USP473.8kb

New Vector Acquires Higher Virus Titer

Vector pLAS_AS3w.Ppuro pLAS_AS3wd.Ppuro

USP473.8kb

CAG p puro

AS3w.PpurohPGKp

1026bp

shRNA Cloning: Strategy & Troubleshooting

target sequence siRNA sequenceccggcgcatacgacgattctgtgatctcgagatcacagaatcgtcgtatgcgttttt

ctcgag: loop sequence of shRNA

gcgtatgctgctaagacactagagctctagtgtcttagcagcatacgcaaaaattaa

shRNA sequence design: for annealing method

AgeI end EcoRI end

Annealing parameters:950C, 780C, 740C, 700C, 670C, 630C, 600C, 560C, 630C, 600C, 560C, 530C, 500C, 480C, 460C, 440C, 420C, 400C, 390C, 370C, 360C, 350C, 340C, 330C, 320C, 310C----------------------------------------------5 min for each setting300C, 280C, 260C, 240C, 220C, 200C---------------10 min for each settingHold at 40C

10X annealing buffer:1M K-acetate0.3M HEPES-KOH pH7.420 mM Mg-acetate

Setting up annealing mixture:100 mM sense oligo 9 ml100 mM antisense oligo 9 ml10X annealing buffer 2 ml

Annealing Conditions

shRNA sequence design: for PCR method

55O C

59O C

64O C

66O C

69O C

20 cyclesTemplate: I ml of 1 mM

M 1 2 3 4

3 Cyc

les

5 Cyc

les

8 Cyc

les

10 C

ycle

s

Template: I ml of 50 mM

Optimization of the PCR Conditions for PCR-Based shRNA Construction

500bp

300bp

100bp

M M 1 2 3 4

63bp

1. PCR products (5 cycles)

2. PCR cleaning

3. BsmBI digestion (before purification)

4. Column purified products (after digestion)

Preparation of PCR Products for shRNA Cloning

500bp

300bp

100bp

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

3 cycles (PCR)

+ 3

cycl

es+

2+

3

Synthesized oligos have different amplification efficiency

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1. LKO_TRC0012. LKO_TRC0053. LKO_TRC0074. LKO_TRC0085. LKO_TRC009

6. LKO_TRC0107. LKO_TRC0118. LKO_TRC0139. LKO_TRC01410.LKO_TRC016

11. LKO_TRC01712. LKO_TRC01813. LKO_TRC01914. LKO_TRC02015. LKO_TRC024

9.0kb

3.0kb

2.0kb1.5kb

Digestion Pattern of LKO_TRC shRNA VectorsAgeI/EcoRI double digestion

Stem-Loop Structure of shRNA May InterfereDNA Sequencing

Without resolution buffer:

With resolution buffer:

Virus Titer: Effect of DNA-Prep

pDNA Preparation Method Titer(RIU/uL) STDEV Sample

No.

Geneaid High-Speed Mini 24,371 1525 5

Qiagen Midi 22,385 2313 16

Viogene Midi Plus Ultrapure 19,685 4252 11

PEG 18,747 519 3

Invitrogen-HiPure Midi 15,937 3160 22

NUCLEObond AX 15,007 2852 3QIAprep spin (Phenol/chloroform) 14,210 2296 7

Qiagen Mini 13,732 1850 8

Off-target of siRNA

Nature Review5: 522, 2004

Degradation of mRNA can occur by two separate pathways in RNAi

Seed Sequence

3’ UTR hexamer frequency in human genome

SCF: seed complementary frequency

high(>3800), medium (z2500–2800), or low (<350) SCFs in the HeLa transcriptome

Khvorova A. RNA (2008),14:853-861.

Microarray signatures of GAPDH- and PPIB-targeting siRNAs

Same seed sequences in different target genes:GAPDH H15 sense: 5-GAAGUAUGACAACAGCCUC PPIB H17 sense: 5-CGACAGUCAAGACAGCCUG

One nt shift in seed sequence: GAPDH M1 sense: 5-GGCUCACAACGGGAAGCUU

GAPDH M8 sense: 5-GCUCACAACGGGAAGCUUGSeed region not static

Khvorova A. RNA (2008),14:853-861.

Anderson E. M. et.al. RNA;2008;14:853-861

GAPDH PPIB

Off-Target Numbers of GAPDH- and PPIB-targeting

siRNAs

Seed sequence plays major role in off-target

GAPDH

high(>3800), medium (z2500–2800), or low (<350) SCFsin the HeLa transcriptome (z10 siRNAs for each group) Khvorova A. RNA (2008),14:853-861.

Configuration of TRC shRNA construct

How are the TRC library shRNAs processed into short dsRNAs?

Implications: hairpin design, off-target effects

A

CGGGTCGAGCTGGACGGCGACGTACT

GTTTTTCAGCTCGACCTGCCGCTGCATG

Which strand goes into RISC?(Strand that goes into RISC is more stable/abundant)

Where does DICER cut?polIII transcription start and stop;evidence for DROSHA processing?

shRNA processing

TRC: Jen Grenier, Andrew Grimson, Ozan Alkan

22 nts

22mer 18,285 4% 5% 21mer 39,095 9% 10%20mer 6,760 2% 2%

23mer 45,610 10% 11%22mer 205,249 46% 51%21mer 40,444 9% 10%

23mer 23,263 5% 6%

r4Tsr5Ts

GGGGGG

23mer 5,217 1% 11%22mer 32,279 7% 67%21mer 8,029 2% 17%20mer 1,029 <1% 2%

GG21merSenseStrandSeqncC

G21merAntisenseStrandSTTTTTA

TC

G

Length#reads % shRNA % strand

r

ee

3Ts

5Ts4Ts

GGe3Ts

m4Ts

} 17%

72%

(5)

(3)

(4)

Small RNA sequencing: all 26 shRNAs

Done by Solexa sequencer

Configuration of siRNA Dual-Luciferase Reporter System

: Polyadenylation signal sequence

List of All Possible Seed Complement Frequency Hexamer as an Example

Common feature of SCF: Sequence with CGCG.

Knockdown Measurement of Scramble shRNAs

R/F Lu

c

R/F Lu

c #1

R/F Lu

c #2

R/F Lu

c #3

R/F Lu

c #4

R/F T #1

R/F T #2

R/F T #3

R/F T #4

0

20

40

60

80

100

120 Remaining R/L

Construct

Rela

tive

R/L

(%)

Knockdown Measurement of Scramble shRNAs

55’’--GGCAAATCACAGAGGCAAATCACAGAATCGTCATCGTCGTAGTA--33’’

33’’--TTTTGTTTAGTGTCTTTTTGTTTAGTGTCTTAGCAGTAGCAGCC--55’’

Distribution of Blast Hits on the Distribution of Blast Hits on the shLuc221 Sequence shLuc221 Sequence

55’’--GGACTAGAGTTTCGGACTAGAGTTTCCGCGTACGCGTAAATAAT

33’’-- TTCCTGATCTCAAAGTTCCTGATCTCAAAGGCGCATGCGCATTT --55’’

Distribution of Blast Hits on the Distribution of Blast Hits on the Scramble 1 Sequence Scramble 1 Sequence

55’’--GGAGTTCAGTTACTCGCGAATG --33’’

33’’--TTTTTCAAGTCAATGTTTTTCAAGTCAATGAGCGCTAGCGCTTT--55’’


55’’--GGAGTTCGATATCACGCGATAC --33’’

33’’--TTTTTCAAGCTATAGTTTTTCAAGCTATAGTGCGCTTGCGCTAA--55’’


55’’--GGACTAGAGTTTCTGCTTTAAT --33’’

33’’--TTTTTGATCTCAAAGTTTTTGATCTCAAAGACGAAAACGAAATT--55’’

Distribution of Blast Hits on the Distribution of Blast Hits on the Scramble m1 Sequence Scramble m1 Sequence

55’’--GGACTAGAGTTTCTGCTTTAAT --33’’

33’’--TTTTTGATCTCAAAGTTTTTGATCTCAAAGACGAAAACGAAATT--55’’


55’’--GGAGTTCAGTTACGATATCATG--33’’

33’’--TTTTTCAAGTCAATGTTTTTCAAGTCAATGCTATAGCTATAGTT--55’’


Documents

1 PI: Dr. James C.-K. Shen ( 沈哲鯤 ) Co-PI: Dr. Michael Hsiao ( 蕭宏昇副研究員 ) Co-PI/ Manager: Dr. King-Song Jeng ( 鄭金松副技師 ) Speaker: King-Song Jeng ( 鄭金松