Supporting Information
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Supporting Information Fei et al. 10.1073/pnas.1617467114 SI Materials and Methods Cont. Cell Culture, Reagents, and Antibodies. LNCaP, CWR22Rv1, DU145, Gene name siRNA-a siRNA-b and PC3 cells were cultured in RPMI medium 1640 supplemented GUAGUAGUGUCUUACUGGU GUUGGGUCCCUCUGAAGUU with 10% FBS. RWPE-1 cells were cultured in K-SFM (Kit Cat. no. HNRNPD CUUAGUAAGCUGGUCCAGA CUAUAUGGAUGAGGUGACA 17005-042) with kit-supplied bovine pituitary extract and human HNRNPUL2 CUCAUGUAACUGUGAAGAA CUGAUAGGCAGUCUGGAAA recombinant epidermal growth factor. RNase R was purchased HNRNPA2B1 HNRNPLL AGUGCAACGUAUUGUUAUA CUUAAUGUUUGCGUGUCUA from Epicentre (Cat. no. RNR07250). The antibodies were pur- HNRNPA1 GGAAGAGUUGUGGAACCAA GUGGUAAUGAGAGAUCCAA chased from the following sources: GAPDH (FL-335, Santa Cruz, HNRNPR GGAGUAUGGAGUAUGCUGU GCUAGUGCUUUGUCUUAGU Cat. no. sc-25778), HNRNPL (4D11, Santa Cruz, Cat. no. sc-32317; HNRNPAB GAGAUUGAGGCCAUUGAAU CUGUGGACCCUGGUUGUAA D5, Santa Cruz, Cat. no. sc-48391), HNRNPK (3C2, Santa Cruz, Cat. no. sc-32307), and AR (N-20, Santa Cruz, Cat. no. sc-816). RNA Isolation and qRT-PCR. RNA was isolated using the RNeasy Pooled Genome-Wide CRISPR Screen. LNCaP cells were cultured in Mini Kit (Qiagen). Reverse transcriptase (Invitrogen) was used RPMI medium 1640 supplemented with 10% FBS and infected with for random-primed first-strand cDNA (cDNA) synthesis. Real- the pooled lentiviral library (GeCKO v2 library) at an multiplicity of time PCR was carried out on ABI Prism 7300 detection system infection of 0.5. Large-scale spin-infection of 1 × 108 cells were usingSYBRGreenPCRmastermix.TheΔΔCt method was carried out in four 12-well plates with 2 × 106 cells per well. Wells used to comparatively quantify the amount of mRNA level. were pooled together into larger flasks on the day after spin- RPS28 gene expression served as the internal control. Primer infection. After 3 d of puromycin selection, half of the surviving sequences detecting mRNA levels are listed below: cells were stored as 0-d control samples, and the rest of the cells were cultured in white RPMI medium 1640 supplemented with Gene name Forward primer (5′ to 3′) Reverse primer (5′ to 3′) 10% charcoal-stripped FBS in the presence of 10 nM DHT for an additional 2 wk. PCR was performed on genomic DNA to con- RPS28 CGATCCATCATCCGCAATG AGCCAAGCTCAGCGCAAC AGCTCCCGCTCGAATCTGAT CCTCAACTCGCAGTCAAAGTC struct sequencing libraries with each containing around 300 μg HNRNPK ∼ HNRNPL TTCTGCTTATATGGCAATG- GACTGACCAGGCATGATGG DNA each. Each library was sequenced at around 30 40 million TGG reads to achieve ∼300× average coverage over the CRISPR library. HNRNPC GGAGATGTACGGGTCAGTA- CCCGAGCAATAGGAGGAGGA Data analysis was performed by MAGeCK and MAGeCK-VISPR. ACA RBMX GCTCTTCATTGGTGGGCTTA GGGCTTTCAAAGGTGACAAA Targeted siRNA Knockdown for Functional HNRNP Genes. LNCaP HNRNPDL TGTGAGATCACCCGTTGTGT CAGGTTTCAGAGGACCTGGA cells were seeded in a 24-well plate and transfected with 20 nM PCBP1 AAAGGCGGGTGTAAGATCA- GGCAAATCTGCTTGACACACTC siRNA oligos by RNAiMax reagent (Life Technology, Cat. no. AAG 13778-150). Knockdown efficiency was determined after 72 h of HNRNPM TGGTCCGAGCAGACATTC- TGACGTGCATTGGTCTATCAAA transfection. Cell counting was performed after 6 d of transfection. TTG The siRNA oligos targeting the HNRNP genes were purchased FUS TCAATCCTCCATGAGTAG- CACGGTCCTGCTGTCCATA from Sigma and Dharmacon. The siRNA target sequence for TGGT siControl is 5′- GCGACCAACGCCUUGAUUG-3′.ThesiRNA PCBP2 GCGCAGATCAAAATTGCG- ATATTGAGCCAGGCTAATGCTG target sequences for the HNRNP genes are as follows: AAC HNRNPU GAGCATCCTATGGTGTGT- TGACCAGCCAATACGAACTTC CAAA Gene name siRNA-a siRNA-b HNRNPUL1 GAAGCACCTTCCGTCTAC- AGGAGAAAGGCTCTTCGCCTA HNRNPK GCAAGAAUAUUAAGGCUCU GAUCUUGGUGGACCUAUUA AGA HNRNPL CAUCAUGCCUGGUCAGUCA AGGUUUGUAGAGGCUUACU HNRNPF CTGCTCTGTTGAGGACGTG CCTGCCCTCTCTAGTGTAGATG HNRNPC CAGUAGAGAUGAAGAAUGA GAUGAAGAAUGAUAAGUCA SYNCRIP GAGCTAGAGGAAGGGGTGGT CTCTTTGTTGTTGGGCACCT RBMX CGAUAGAGAUGGAUAUGGU CUACUCAAGUGGUCGUGAU HNRNPH3 AATGGTCCAAATGACGCTA- CTCCCCTGGTAGTCCATCGT HNRNPDL GUCACUAUGGAGGAUAUGA CAAGGAUAUGGAAAUUACA GTG PCBP1 CGGUUAAGAGGAUCCGCGA GUAUUAGUCUGGCCCAGUA PTBP1 AGCGCGTGAAGATCCTGTTC CAGGGGTGAGTTGCCGTAG HNRNPM GAUUGACGUUCGAAUUGAU CGAUUUGGAUCUGGGAUGA HNRNPH2 GAAGCATACAGGTCCGAAT- CGCCCCTGAAAGTCCACTG FUS CAGAGCUCCCAAUCGUCUU GGCUAUGGAACUCAGUCAA AGC PCBP2 GCAUUAGCCUGGCUCAAUA GAACCCAGUGGAAGGAUCU HNRNPA3 TGATGGGCGTGTAGTGGAAC AGCAGACTGCATCTCTTGTT- HNRNPU GUGGAAUCGGCUAUCCAUA GUCACUAACUACAAGUGGA TAG HNRNPUL1 CUAUAUCCUAGAUCAGACA GUUGCUAUUGACACCUAUA HNRNPA0 TGGCTTCGTGACCTACTCCAA GGCCTCCGACAAAGAGCTT HNRNPF GGUACAUUGGCAUCGUGAA CAAUAUGCAGCACAGAUAU RALY TTCAGGCAAGCAATGTAACCA CACGGCCATACTTAGAGAAG- SYNCRIP GCUAGUUGCACAUAGUGAU GUUAUGCGUUUGUCACUUU ATG HNRNPH3 GACAGUACGACUUCGUGGA CAAUUACAGUGGAGGAUAU HNRNPH1 ATTCAAAATGGGGCTCAAG- GTGTCAGGACTATTTGGACCAG PTBP1 CAAGAACUUCCAGAACAUA CUGACCAAGGACUACGGCA GTAT HNRNPH2 GUACAUUUGUGGGAGUUGA CUGUACAUUUGUGGGAGUU HNRNPD GCGTGGGTTCTGCTTTATT- TTGCTGATATTGTTCCTTCG- HNRNPA3 GUACAUUCCUGAGGUCUUU CAAUGUGUGCUCGACCACA ACC ACA HNRNPA0 CAGACCAAGCGCUCCCGUU CACUUUGAGGCCUUUGGGA HNRNPUL2 GGCAAAGGTAACCCAGAAT- GGACGGGAAAAATCAACAGACC RALY GGCAAGCAAUGUAACCAAC GCAAGCAAUGUAACCAACA CTC HNRNPH1 CUUCUUGAAUUCUACAGCA CUUUGUACGGCUUAGAGGA HNRNPA2B1 AGCTTTGAAACCACAGAAGAA TTGATCTTTTGCTTGCAGGA Fei et al. www.pnas.org/cgi/content/short/1617467114 1of10 Cont. EGFP: 5′- GATCACAATTAACCCTCACTAAAGGGATG- GTGAGCAAGGGCGAGGAGC-3′ and 5′- GATCACTAA- Gene name Forward primer (5′ to 3′) Reverse primer (5′ to 3′) TACGACTCACTATAGGGTTATCTAGATCCGGTGGAT- HNRNPLL ACCATTCCTGGTACAGCACTG TGGCCAGCACTTGTAAAGC CCC-3′; TCAGAGTCTCCTAAAGAGCCC ACCTTGTGTGGCCTTGCAT HNRNPA1 RPS28: 5′- GATCACAATTAACCCTCACTAAAGGGCCA- GCAAGGTGCAAGAGTCCACA CACGCCAGAGTACACACTGTC HNRNPR TCATGGACACCAGCCGTGTG-3′ and 5′- GATCACTAA- ATTGAGGCCATTGAATTGCCA GGCCACCTTGATCTCACACTT HNRNPAB TACGACTCACTATAGGGAACTTGAAACACAAACGC- TGTGTGCTGGACGCTGGA CACTGCCCCATGACGTGAT KLK3 TTTAT-3′; TMPRSS2 GGACAGTGTGCACCTCAAAGAC TCCCACGAGGAAGGTCCC FKBP5 GCGGAGAGTGACGGAGTC TGGGGCTTTCTTCATTGTTC LARP: 5′- GATCACAATTAACCCTCACTAAAGGGCCT- GGTGACTCGGACATTCCAGG-3′ and 5′- GATCACTAA- TACGACTCACTATAGGGTGATCCGCTGTGCGGCCA- RIP. Adherent cells grown in 15-cm plates were first cross-linked CAGGTC-3′; with 0.3% formaldehyde for 10 min at room temperature before the reaction was quenched by adding one-tenth volume of 1.25 M CTBP1: 5′- GATCACAATTAACCCTCACTAAAGGGCA- glycine for 5 min. Cells were then scraped off the plates and lysed GATAACGTACACGGATGCCACAG-3′ and 5′- GATCAC- with RIPA lysis buffer (50 mM Tris, pH7.6, 150 mM NaCl, 1 mM TAATACGACTCACTATAGGGGTGTGTGACATCTGT- EDTA, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, GCAGGCCCTG-3′; protease inhibitor, and RNase inhibitor) for 10 min on ice. Then ROR2: 5′- GATCACAATTAACCCTCACTAAAGGGACC- the lysate was sonicated to assist solubilization and RNA frag- TTCTTACTGCCCCTTCTTCTTC-3′ and 5′- GATCACTA- mentation before centrifugation at 20,000 × g for 10 min at 4 °C. ATACGACTCACTATAGGGTCTTTGTGTGTGTCTGAA- The supernatant was collected and precleared with protein G TATTCTG-3′; beads. The input fraction was obtained from the supernatant after preclear step. The antibodies were preincubated with pro- STX3: 5′- GATCACAATTAACCCTCACTAAAGGGTTG- tein G beads and washed with RIP wash buffer (RIPA lysis TAGGAATTGTGTCTGGAACC-3′ and 5′- GATCACTAA- buffer without inhibitors) before adding the precleared cell ly- TACGACTCACTATAGGGACAGCTCTCTGATATATCA- sate. After 4- to 6-h incubation, the beads were washed twice AATTCC-3′. with RIPA lysis buffer followed by three washes with 1 M RIPA buffer (1 M NaCl in RIPA lysis buffer). The RNA was eluted Tissue Microarray Analysis. The use of human prostate samples from the beads with 100 μL NaHCO and 1% SDS in the has been approved by The Gelb Center Committee. Immuno- 3 μ presence of proteinase K and RNase inhibitor at room temper- histochemical staining for HNRNPL was performed on 4- m ature for 10 min with occasional vortexing. The eluted material paraffin sections cut from two prostate tissue microarray (TMA) was decross-linked at 65 °C for 45–60 min before purification of blocks provided by the Gelb Center Tissue Bank. The immuno- RNA using TRIzol LS reagent (Life Technology). DNase I chemical stain was initially optimized on the bench, and then treatment was performed to remove any residual DNA before transitioned to the Leica BOND-III (Leica Biosystems) autostain- phenol/chloroform/ethanol purification of the final RNA. RIP ing system. Immunostaining was performed on tissue sections fol- RNA can be used for either library preparation or direct qPCR lowing deparaffinization in two 5-min changes of xylene and assay. HNRNPL (4D11) antibody was used for HNRNPL RIP- rehydration through graded alcohols to distilled water. After seq. The RIP-qPCR primers used in this study are as follows: blocking endogenous peroxidase activity, sections were sub- jected to heat-induced epitope retrieval in citrate buffer (pH 6.1) for 30 min. Following heat-induced epitope retrieval, the primary Gene name Forward primer (5′ to 3′) Reverse primer (5′ to 3′) mouse monoclonal antibody targeting HNRNPL (4D11, sc- RPS28 CGTGGAATTCATGGACGAC GCTTCTCGCTCTGACTCCAA 32317, Santa Cruz Biotechnology) was applied to the sections. CTBP1 ACGTCTGTGCTGTGATGTCC CGGATGTCATAGATGCCACA Protein levels were examined using a dilution of 1:5,000 for 1 h at ROR2 GTGTCATTCAATATTCTGT- ACAGAGAACACACTTAGAGA- room temperature. Incubation with the biotinylated universal sec- GTGTG CACAA ondary antibody was then performed. Visualization was performed STX3 GCCATGTTTTAGCTGTGTGG TTGTTGCTGTTGGTTGTGGT