GSM9321003: U2OS_HA_eCLIP_CBL0137_RBRIIIMUT_R2; Homo sapiens; OTHER
seCLIP (single-end enhanced cross-linking and immunoprecipitation) sequencing was performed as described in Van Nostrand et al. and Blue et al. All seCLIP experiments were conducted in duplicates with one pooled size-matched inputs per condition. Per IP sample, 30-40 million cells were crosslinked with UV-C light (254 nm, 2 min, 400 mJ/ cm2). Not UV-crosslinked cells were used as negative control. Cells were lysed in seCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% (vol/vol) Igepal CA-630, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and 440 U/ml murine RNase inhibitor (NEB), and 10% of crosslinked murine NIH 3T3 cells were added to each sample as spike-in (*). Samples were sonicated using the M220 Focused-ultrasonicator (Covaris) for 3 min. Next, samples were treated with 40 U RNase I (Thermo Fisher Scientific) and 10 U Turbo DNase for 5 min at 37  °C shaking at 1200 rpm. After centrifugation at 15,000 g for 10 min at 4 °C, supernatants were added to pre-coupled protein A/G Dynabeads (62.5 µl each per IP) to the antibody of interest (10 µg per sample) and incubated rotating at 4 °C overnight. 2% v/v of the lysate (20 µl) including beads were taken as SMInput, both for the RNA gel and for the diagnostic western blot. The remaining lysate was magnetically separated and washed 2 times with high-salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1% (vol/vol) Igepal CA-630, 1 mM EDTA, 0.1% (vol/vol) SDS and 0.5% (wt/vol) sodium deoxycholate), followed by a transition wash which was conducted by adding 500 µl of high-salt wash buffer, mixing and then adding 500 µl of wash buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% (vol/vol) Tween-20 and 5 mM NaCl). Three more washes with wash buffer were conducted, before the immunoprecipitated RNA was dephosphorylated on-beads using the FastAP and PNK enzymes. Therefore, 1X FastAP buffer (Thermo Fisher Scientific), 80 U murine RNase inhibitor (NEB), 4 U Turbo DNase (NEB) and 3 U FastAP enzyme (Thermo Fisher Scientific) were added in a total volume of 50 µl and incubated for 20 min at 37 °C shaking at 1,200 rpm. Without removing the phosphorylation mix, 150 µl PNK master mix (1x PNK7 buffer (700 mM Tris-HCl pH 7, 100 mM MgCl2), 40 U PNK4 enzyme) were added and incubated for another 20 min at 37 °C (shaking at 1,200 rpm). Samples were then washed with 200 µl seCLIP high-salt wash buffer once, followed by a transition wash and three further washings with seCLIP wash buffer. 10% of the IP samples were saved for biotin-labeling to visualize the RNA cross-linked to the protein of interest. To the remaining RNA, InvRiL19 RNA adapters (IDT) were ligated by incubating the samples with 25 µl 3'RNA linker mix (1.2x RNA ligase buffer (no DTT), 1.2 µM ATP, 3.6% DMSO, 0.024% v/v Tween-20, 18% w/v PEG 8000, 0.8 U murine RNase inhibitor (NEB), 72 U T4 RNA ligase high-concentration enzyme) rotating at RT for 75 min. Beads were then washed as follows: 1x wash buffer, transition wash to high-salt wash buffer, 1x high-salt wash buffer, transition wash to wash buffer, 3x wash buffer. After the final wash, 20% per IP sample was kept for the diagnostic western blot. To all IP and SMInput samples, NuPAGE LDS buffer (Thermo Fisher Scientific) containing DTT (98 mM, freshly prepared) was added before samples were incubated at 70 °C for 10 min. After magnetically separating the beads from the lysates, RNA-protein complexes were separated by gel electrophoresis using 8% Bis-Tris gels, and transferred onto nitrocellulose or methanol-activated PVDF membranes for the RNA gels and western blots, respectively. The diagnostic western blot was used as a guide to cut out the region of the membrane starting from the observed size of the protein of interest and extending until the maximal expected size of the RNA-protein complexes (but at least ~75 kDa larger). Membrane pieces were then sliced into ~2 mm x 2 mm squares and transferred into a fresh tube. To digest the protein, membranes were incubated with 24 U proteinase K (Roth) in PKS buffer for 20 min at 37 °C followed by an incubation at 50 °C for another 20 min. RNA was extracted using the RNA Clean and Concentrator-5 kit (Zymo) following the manufacturer's instructions, and RNA was eluted in 10 µl H2O. Next, SMInput samples were dephosphorylated by incubating each SMInput RNA sample with 20 µl of dephosphorylation master mix (1x FastAP buffer (Thermo Fisher Scientific), 40 U murine RNase inhibitor (NEB), 2 U FastAP enzyme (Thermo Fisher Scientific) incubating at 37 °C for 20 min shaking at 1,200 rpm, followed by another 20 min incubation at 37 °C (1,200 rpm) in PNK master mix (1.05x PNK7 buffer, 5.26 mM DTT, 2 U Turbo DNase (NEB), 40 U T4 PNK enzyme). RNA was purified using the Clean & Concentrator 5 kit (Zymo) as described before. To append the SMInput RNA with 3' RNA adapters, 1.5 µl 100% DMSO and 0.5 µl InvRil19 adapter were added per sample and incubated at 65 °C for 2 min. 13.5 µl of SMInput RNA ligation master mix (0.976x RNA ligase buffer (NEB), 0.976 mM ATP, 2.93% DMSO, 0.02% v/v Tween-20, 14.63 w/v PEG 8000, 12 U murine RNase inhibitor, 36 U T4 RNA ligase high-concentration enzyme) were added and samples were incubated at RT for 60 min on a rotating wheel. To purify the RNA, 15 µl MyOne Silane Dynabeads per sample were washed ones with RLT buffer (Qiagen), resuspended in RLTW buffer (RLT + 0.025% v/v Tween-20) and added to the RNA. An equal volume of 100% EtOH was added and samples were incubated at RT for 10 min, before samples were washed three times with 80% v/v EtOH. After the final wash, beads were tried and subsequently eluted in 9.5 µl of TT elution buffer (10 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.01% (vol/vol) Tween-20). All IP and SMInput samples were next reverse transcribed using the SuperScript III enzyme. For that, 1 µl of 5 µM InvAR17 RT primer  and 1 µl of 10 mM dNTPs were added per RNA sample and incubated for 2 min at 65 °C. 10 µl of reverse transcription master mix (1x first strand buffer (Invitrogen), 5 mM DTT, 8 U murine RNase inhibitor (NEB), 120 U Superscript III enzyme (Invitrogen)) were added to each sample and incubated at 55 °C for 20 min. 2.5 µl ExoSAP-IT (Thermo Fisher Scientific) were used to remove unincorporated dNTPs and RT primers (37 °C, 15 min) before 1 µl of 0.5 M EDTA was added. To remove RNA stands, 3 µl of 1 M sodium hydroxide were added and incubated at 70 °C for 10 min. 3 µl of 1 M hydrochloric acid were added to neutralize the sodium hydroxide. RNAs were again purified using MyOne Silane beads as described earlier. After drying, 2.5 µl of 5' adapter master mix (4.66 µM InvRand3Tr3 adapter and 7.77% DMSO in TT elution buffer) were directly added to the beads and incubated at 70 °C for 2 min before 7.8 µl of ligation master mix (0.097% RNA ligase buffer (NEB), 1.94 mM DTT, 0.97 mM ATP, 0.019% v/v Tween-20, 17.48% PEG 8000, 30 U T4 RNA ligase high-concentration enzyme (NEB), 5' deadenylase enzyme (NEB)) and incubated at RT overnight. RNA was subsequently purified using MyONE silane beads as described earlier. To estimate the number of cycles required for the final PCR, qPCRs were conducted. For multiplexing of eCLIPs, each RNA adapters containing unique barcodes (RNA_A01, RNA_B06, RNA_C01, RNA_D08, RNA_X1A, RNA_X1B) based on 44 were ligated to the RNA after immunoprecipitation. At least one third of each sample was mixed and further processed as a pool. SMI were separately run through the gel and barcoded RNA adapters were ligated after RNA isolation from the nitrocellulose membranes. As for the IP samples, inputs were merged to a pool and processed together. For total input samples, 20 µl of lysate were taken before the immunoprecipitation. Using the eCLIP adapter strategy 44, the in-line barcode on read 1 was later used to demultiplex the samples bioinformatically. Samples labeled with * contain 10% murine spike-in (NIH 3T3) that has not been used for further analysis. For fractionation eCLIPs (+) cells were cross-linked as described above. Cells were then fractionated as described in Nojima et al. Briefly, cells were resuspended in ice-cold HLB+N buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2 and 0.5% (vol/vol) NP-40) and incubated for 5 min on ice. The lysates were then resuspended in 1 ml ice-cold HLB-NS buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 2.5 mM MgCl2, 0.5% (vol/vol) NP-40 and 10% (wt/vol) sucrose) and centrifuged at 420 g for 5 min. The cytosolic fraction in the supernatant was removed and the nuclear pellet was resuspended in ice-cold NUN1 buffer (20 mM Tris-HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, and 50% (vol/vol) glycerol), followed by the addition of ice-cold NUN2+E buffer (20 mM HEPES-KOH (pH 7.6), 300 mM NaCl, 0.2 mM EDTA, 7.5 mM MgCl2, 1% (vol/vol) NP-40, 1 M urea, and 3% Empigen (Sigma, Cat. No. 30326)). After incubation for 5 min on ice, samples were centrifuged at 300 g for 1 min at 4°C and the nucleoplasmic supernatant was separated from the chromatin pellet. The chromatin pellet was washed twice with ice-cold PBS. Immunoprecipitations were performed in seCLIP Lysis Buffer. All subsequent steps were performed as described for eCLIP multiplex sequencing. cDNA libraries were amplified using a three-step, six cycle amplification followed by a two-step amplification bringing each sample to its calculated number of cycles. The final libraries were either purified using an agarose gel-based strategy cutting out gel pieces corresponding to 175-300 bp in size and purifying the DNA using the Qiagen MiniElute gel extraction kit following the manufacturer's instructions. Alternatively, samples were cleaned using a two-sided selection with SPRI beads (0.7x right-sided, followed by a 1x left-sided selection). The quality and quantity of the libraries were assessed using the Fragment Analyzer (Agilent). To account for different cycle number used during final PCR amplification (e.g. for IgG controls), DNA spike-in containing adaptors for the Illumina index primers used in the final PCR was generated and equal amounts were added to the immunoprecipitation samples prior to the final PCR for indicated experiments (*#), resulting in different relative ratios of CLIP:DNA spike-in in the final libraries as observed upon measurement using the fragment analyzer.