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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.
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 : RNAiCore@imb.sinica.edu.tw
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
21
Chi-long Lin
Navigation of RNAi Website
Navigation of RNAi website
–Overview–Search function–Knockdown information
database–New Online Ordering system–Feedback mechanism
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
Navigation of RNAi website
–Overview–Search function–Knockdown information
database–New Online Ordering system–Feedback mechanism
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
Navigation of RNAi website
–Overview–Search function–Knockdown information
database–New Online Ordering system–Feedback mechanism
Navigation of RNAi website
–Overview–Search function–Knockdown information
database–New Online Ordering system–Feedback mechanism
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’’
Distribution of Blast Hits on the Distribution of Blast Hits on the Scramble 2 Sequence Scramble 2 Sequence
55’’--GGAGTTCGATATCACGCGATAC --33’’
33’’--TTTTTCAAGCTATAGTTTTTCAAGCTATAGTGCGCTTGCGCTAA--55’’
Distribution of Blast Hits on the Distribution of Blast Hits on the Scramble 4 Sequence Scramble 4 Sequence
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’’
Distribution of Blast Hits on the Distribution of Blast Hits on the Scramble m1 Sequence Scramble m1 Sequence
55’’--GGAGTTCAGTTACGATATCATG--33’’
33’’--TTTTTCAAGTCAATGTTTTTCAAGTCAATGCTATAGCTATAGTT--55’’
Distribution of Blast Hits on the Distribution of Blast Hits on the Scramble m2 Sequence Scramble m2 Sequence
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