1. 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. 2 Outline The RNAi Consortium (TRC) The RNAi Consortium (TRC) Current status of the RNAi Core Current status of the RNAi Core Collaboration Research Project Collaboration Research Project

3. 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. 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. 5 Sponsoring MembersCollaborating 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. 6 Lentivirus-based RNAi (VSV-G peudotyped virus) http://www.accessexcellence.org/RC/VL/GG/retrovirus.html shRNA

7. 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)

8. TRC2 library goals More powerful library: – enriched for best OT KD shRNAs – even coverage of genome TRC2 library 3+ good shRNAs per gene High-performing existing clones New clones(test)

9. 9 Materials Received from TRC shRNA constructs and knockdown information: TRC-ITRC-II Clone #Gene #KDClone #Gene #KD Human81,88816,02634,89346,4285,5165,040 Mouse77,70015,97632,18831,2024,3891,753 Control85129 Total159,58832,00267,08177,7599,9056,793 Pooled genome-wide shRNA plasmid DNAs and chips (human and mouse) for RNAi genome-wide screening.

10. 10 67,000 TRC shRNAs targeting 12,200 genes (Jurkat data excluded) J. Grenier All Hairpins StatisticsPer Gene Statistics TRC shRNA performance stats: Sept.09

11. How reproducible are the data?: How consistent is the > 70% call? Trial 1 Trial 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. 12 TRC TAIWAN RNAiCORE

13. 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. 14 Aims of RNAi Core: Third Phase Objectives: Maximizing the utilization of the RNAi library in mammalian genetic screen

15. 15 Routine Service Items Categories Categories Service Items Service Items Bacterial Clones Bacterial Clones shRNA construct shRNA construct VSV-G lentivirus VSV-G lentivirus Arrayed VSV-G pseudotyped lentivirus Arrayed VSV-G pseudotyped lentivirus Pooled VSV-G pseudotyped lentivirus Pooled VSV-G pseudotyped lentivirus Individual VSV-G pseudotyped lentivirus Individual VSV-G pseudotyped lentivirus Customized lentivirus Customized lentivirus HT&HCS Image Analysis HT&HCS Image Analysis Plasmid DNA Plasmid DNA (lentiviral transfer vector) Package plasmids Package plasmids shRNA cloning lentivector shRNA cloning lentivector Pol II/ gene expression lentivector * Pol II/ gene expression lentivector *

16. 16 Analyses of Geographic Distribution and Distributed Items to the Users Total Users: 465 PIs Bacterium: 79% Virus: 6% HTC image analysis: 2% Lentiviralvector: 13% 2% 79% 6% 13% Total Users: 465 PIs 11% 58% 3% 20% 8% AcademiaSinica(AS): 11% N.Taiwan(ASnot included): 58% Middle Taiwan: 8% Southern Taiwan: 20% Eastern Taiwan: 3%

17. 17 User Publication 25 5 4 0 2 4 6 8 10 12 14 16 18 20 <55~10>10 Impact Factor # of publication 6 16 0 2 4 6 8 10 12 14 16 18 20 2006~20072008~2009 Year # of publication 12 2009~2010 Please acknowledge the RNAi Core for RNAi reagents when your research is get published. Please refer to http://rnai.genmed.sinica.edu.tw/faq-detail.asp?sn=16 http://rnai.genmed.sinica.edu.tw/faq-detail.asp?sn=16 for example.

18. 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 C5: perform microarray and data analysis C6: provide pooled virus C6: provide pooled virus

19. 19 Collaborative Research Project(s) workflow and regulations Current regulations: Tying with the workload of the Core ; Tying with the workload of the Core ; Sharing cost and labor ; Sharing cost and labor ; Yes No Reject or Revise Yes Submission Re-submission RNAi screen and data analysis Data arrangement and release Discuss on the Potential project Review by committee Case closed Notify user committee Future capacity Could accommodate more projects Could accommodate more projects if transform Core into National RNAi if transform Core into National RNAi Screening Center (NRSC). Screening Center (NRSC).

20. 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 RNAiCore@imb.sinica.edu.tw 核心地址 : 台北市南港區 115 研究院路二段 128 號

21. 21

22. Chi-long Lin

23. Navigation of RNAi website –Overview –Search function –Knockdown information database –New Online Ordering system –Feedback mechanism

44. Navigation of RNAi website –Overview –Search function –Knockdown information database –New Online Ordering system –Feedback mechanism

65. Navigation of RNAi website –Overview –Search function –Knockdown information database –New Online Ordering system –Feedback mechanism

66. TRC validation progress: Sept.09 J. Grenier, X. Yang Recent Pace: - Attempts: 1,000 genes/month - Successes: 750 genes/month Cumulative: 67,000 TRC shRNAs Targeting 12,200 genes

74. Navigation of RNAi website –Overview –Search function –Knockdown information database –New Online Ordering system –Feedback mechanism

103. Navigation of RNAi website –Overview –Search function –Knockdown information database –New Online Ordering system –Feedback mechanism

107. Issues Regarding Lentiviral Transfer Vectors and shRNA/siRNA- triggered Off-Target Speaker: 鄭金松, RNAi Core Manager 98/10/16

108. 3’LTR pLKO_AS2 EMCV IRES2 5’LTR puro Maps of lentivirus-based cDNA- expressing transfer vectors CMVie p CAG p Ubiqui p hPGK p EF1  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

109. PCR Amplification of DNA Fragments Sticky-End PCR Method PCR 5’-CTAGC 3’-GATCG 5’-C 3’-G -3’ -5’ -3’ -5’ & Purify and mix 2 PCR products in an equimolar ratio; then de-nature and re-nature 5’-CTAGC -3’ 3’-G -5’ (Effective annealing PCR product for ligation) Primer #1 5’-CTAGC 5’ # Primer #2 Primer #1 5’-CTAGC 5’ # Primer #2 Separated PCR tube #1 Separated PCR tube #2 # Both reactions use the same reverse primer for PCR amplification.

110. (CAGp) (Ubqp)(hPGKp) (EF1  p) Expression Stability of Lentivirus-based System: Effect of Promoters Cell: A549 Wen-Ya

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

112. Genome Length of HIV-1

113. Potential Insertion Length of pLKO_AS3w.puro Vectors HIV 5’-LTR-HIV 3’-LTR = ∼ 5732bp Potential Insertion Size: 9749-5732= 4017bp

114. Genome Organization of HCV

115. 1kbMarker 10k 8k 6k 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

116. 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 5AB Insert IRES Puro-resistant day 2 Puro-resistant day 5

117. HA-NS3-4A NS3-4AB-HA Western Analysis of Lentivirus Transducing Cells Study of the HCV NS Proteins 1 2 3 4 5 Ctl NS4B-HA HA-NS5B NS5A-HA HA-NS3-4A Transient Transfection (done by Ti-Chun) mock NS4B-HA HA-NS5B NS5AB-HA NS5A-HA 1 2 3 4 5 6 7 130 95 75 55 43 34 25 kD Virus Transduction (Done by Huey-Lan)

118.  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

119. Structure of the 5’-UTR of the EMCV IRES

120. Human PGK p Replacement of IRES Element by hPGK Promoter

121. CAG p puro AS3w.Ppuro hPGKp 1.9 3.8 4.4 4.6 0 3.7 +2 +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 5AB 4B 5A 5B 34A 34AB 5AB Insert Puro-resistant d2 IRES sequence may be disrupted by upstream sequences. Upstream Insert Sequences Plays a Role in IRES-directed Translation Puro-resistant d5

122. Secondary Structure of HIV-1 Psi Sequence JMB 326: 529, 2008

123. pLKO.1-puro: Cis Elements That Are Required for HIV-1 Replication

124. NruI 1268bp Can sequences Be Deleted Without Affecting Virus Replication in AS3w.Ppuro Vector RRE = 242bp Potential increase of insert length = 1268bp – 242bp = 1026bp

125. Features of New Lentiviral Transfer Vector HIV 5’-LTR-HIV 3’-LTR = ∼ 4706bp Potential Insertion Size: 9749-4706= 5043bp

126. CAG p puro AS3wd.Ppuro hPGKp 6.9+E5; 3.6+E5 1.5+E6; 1.9+E6 many Insert (kb) Titer Titer Insert Puro resistant cells USP47 3.8kb New Vector Acquires Higher Virus Titer Vector pLAS_AS3w.Ppuro pLAS_AS3wd.Ppuro USP47 3.8kb CAG p puro AS3w.Ppuro hPGKp 1026bp

127. shRNA Cloning: Strategy & Troubleshooting

128. target sequence siRNA sequence ccgg cgcatacgacgattctgtgatctcgagatcacagaatcgtcgtatgcgttttt ctcgag: loop sequence of shRNA gcgtatgctgctaagacacta gagctctagtgtcttagcagcatacgcaaaaa ttaa shRNA sequence design: for annealing method AgeI end EcoRI end

129. Annealing parameters: 95 0 C, 78 0 C, 74 0 C, 70 0 C, 67 0 C, 63 0 C, 60 0 C, 56 0 C, 63 0 C, 60 0 C, 56 0 C, 53 0 C, 50 0 C, 48 0 C, 46 0 C, 44 0 C, 42 0 C, 40 0 C, 39 0 C, 37 0 C, 36 0 C, 35 0 C, 34 0 C, 33 0 C, 32 0 C, 31 0 C----------------------------------------------5 min for each setting 30 0 C, 28 0 C, 26 0 C, 24 0 C, 22 0 C, 20 0 C---------------10 min for each setting Hold at 4 0 C 10X annealing buffer: 1M K-acetate 0.3M HEPES-KOH pH7.4 20 mM Mg-acetate Setting up annealing mixture: 100  M sense oligo 9  l 100  M antisense oligo 9  l 10X annealing buffer 2  l Annealing Conditions

130. shRNA sequence design: for PCR method

131. 55 O C 59 O C 64 O C 66 O C 69 O C 20 cycles Template: I  l of 1  M M 1 2 3 4 3 Cycles 5 Cycles 8 Cycles 10 Cycles Template: I  l of 50  M Optimization of the PCR Conditions for PCR-Based shRNA Construction

132. 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

133. 500bp 300bp 100bp M 1 2 3 4 5 6 7 8 9 10 11 M 2 5 6 3 cycles (PCR) + 3 cycles + 2 + 3 Synthesized oligos have different amplification efficiency

134. Digestion Pattern of LKO_TRC shRNA Vectors AgeI/EcoRI double digestion

135. Stem-Loop Structure of shRNA May Interfere DNA Sequencing Without resolution buffer: With resolution buffer:

136. Virus Titer: Effect of DNA-Prep pDNA Preparation Method Titer (RIU/uL) STDEV Sample No. Geneaid High-Speed Mini24,37115255 Qiagen Midi22,385231316 Viogene Midi Plus Ultrapure19,685425211 PEG18,7475193 Invitrogen-HiPure Midi15,937316022 NUCLEObond AX15,00728523 QIAprep spin (Phenol/chloroform) 14,21022967 Qiagen Mini13,73218508

137. Off-target of siRNA

138. Nature Review 5: 522, 2004 Degradation of mRNA can occur by two separate pathways in RNAi

139. Seed Sequence

140. 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.

141. 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- GGCUCACAACGG GAAGCUU GAPDH M8 sense: 5- GCUCACAACGGG AAGCUUG Seed region not static Khvorova A. RNA (2008),14:853-861.

142. Anderson E. M. et.al. RNA;2008;14:853-861 GAPDH PPIB Off-Target Numbers of GAPDH- and PPIB-targeting siRNAs

143. Seed sequence plays major role in off-target GAPDH high(>3800), medium (z2500–2800), or low (<350) SCFs in the HeLa transcriptome (z10 siRNAs for each group) Khvorova A. RNA (2008),14:853-861.

144. Configuration of TRC shRNA construct

145. How are the TRC library shRNAs processed into short dsRNAs? Implications: hairpin design, off-target effects A C GGGTCGAGCTGGACGGCGACGTAC T G TTTTTCAGCTCGACCTGCCGCTGCATG 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

146. 22mer 18,2854% 5% 21mer 39,0959% 10% 20mer 6,7602% 2% 23mer 45,61010% 11% 22mer 205,24946% 51% 21mer 40,4449%10% 23mer23,2635%6% r 4Ts r5Ts GG 23mer 5,2171% 11% 22mer 32,2797% 67% 21mer 8,0292% 17% 20mer 1,029<1%2% GG21merSenseStrandSeqncC G21merAntisenseStrandSTTTTT A T C G Length#reads% shRNA% strand r e e 3Ts 5Ts 4Ts GG e 3Ts m 4Ts } 17% 72% (5) (3) (4) Small RNA sequencing: all 26 shRNAs Done by Solexa sequencer

147. Configuration of siRNA Dual-Luciferase Reporter System : Polyadenylation signal sequence

148. List of All Possible Seed Complement Frequency Hexamer as an Example Common feature of SCF: Sequence with CGCG.

149. Knockdown Measurement of Scramble shRNAs