GSM8240932: WT_fPAR-CLIP; Homo sapiens; OTHER
Cells were washed once with 1× ice-cold PBS and crosslinked with 0.4J/cm² of 365nm UV light in an Analytik Jena UVLink 3000 Crosslinker (Analytik Jena). Following crosslinking, cells were collected in PBS and centrifuged at 500g at 4°C for 5min. The cell pellet was lysed using the Nuclei EZ following the manufacturer's protocol. The nuclei were then lysed in 3x pellet volume of RIPA buffer supplemented with protease inhibitor (20mM Tris, pH 7.5, 150mM NaCl, 0.1% SDS, 0.15% sodium deoxycholate, 1% NP-40, 0.5mM DTT). The sample was sonicated for 45s (ON time) at 50% amplitude, with 10s ON and 15s OFF, using a Q800R3 Sonicator (Qsonica) and cell extract was clarified at 15,000g, 4°C for 15min. Immunoprecipitation was performed by incubating the clarified extracts with antibodies pre-coated on Protein A&G beads (50µl of 1:1 mixture Dynabead-Protein A and Dynabead-Protein G beads per 0.5 ml of lysate). Binding was performed overnight at 4°C with gentle rotation, and the beads were washed three times with 1 ml of IP buffer (20mM Tris, pH 7.5, 150mM NaCl, 2mM EDTA, 1% NP-40. The beads were treated with 0.15 U/µl RNase I in 2x bead volume of IP buffer at 22°C for 10min. Subsequently, the beads were washed twice with 1ml of each of the following: high salt wash buffer (20mM Tris, pH 7.5, 500 mM NaCl, 2mM EDTA, 1% NP-40), RIPA buffer, and dephosphorylation buffer (50mM Tris–HCl, pH 7.5). On-bead RNA decapping was achieved using Cap-Clip™ Acid Pyrophosphatase (Cellscript, C-CC15011H) according to the manufacturer's instructions, adjusted to 1x beads volume. The reaction was performed at 37°C for 60min with shaking at 1krpm. Beads were then washed twice with dephosphorylation buffer, and on-bead RNA dephosphorylation was achieved using Quick CIP (NEB, M0525S) according to manufacturer's instructions adjusted to 1x bead volume. The reaction was supplemented with 1U/µl RNase inhibitor (SUPERase•In™, Thermo Scientific, AM2694) and performed at 37°C for 10min with shaking. Finally, beads were washed twice with dephosphorylation buffer and twice with ligation buffer without DTT. RNA footprints were ligated to fluorescent 3' barcode adapters (Table S6) using T4 Rnl2(1–249)K227Q (NEB, M0351). A volume of ligation mix (1× T4 RNA Ligase Reaction Buffer, 15% PEG8000, 25µM adapter, 10U/µl Rnl2(1–249) K227Q ligase, 1U/µl RNase inhibitor) equal to that of the beads was added to the beads, and ligation was performed at 4°C for 16h with slow tumbling rotation. The beads were then washed three times with 1ml ligation buffer without DTT. The footprints were phosphorylated using T4 PNK (NEB, M0201S) according to the manufacturer's instructions, adjusted to the volume of the beads. The reaction was supplemented with 1U/µl RNase inhibitor and performed at 37°C for 30min with shaking at 1krpm. The beads were washed twice with high salt buffer and twice with IP buffer. PCF11 was recovered with the addition of 1 bead volume of 2× SDS loading buffer (4x Laemmli Buffer (Bio-Rad  1610747) diluted 2x and supplemented with beta-mercaptoethanol) and incubated at 95°C for 5min. 10% of the eluted RNPs was used for Western blot analysis with an anti-PCF11 antibody. The remaining 90% of the eluted PCF11 was separated on a 4–12% SDS-PAGE alongside 20µl of a 1/10 dilution of Prestained Protein Ladder, 10 to 250 kDa (PageRuler Plus, Thermo Scientific, 26620). The gel was visualized directly using a Bio-Rad imaging system at the IR 660nm detection channel, and the bands corresponding to the PCF11 fluorescent 3′ adapter ligation products were excised and shredded using gel breaker tubes (IST Engineering BioTech, 3388). The shredded gel was incubated with 100µl (1× shredded gel volume) of 2× proteinase K buffer (100mM Tris-HCl, pH 7.5, 150mM NaCl, 12.5mM EDTA, 2% SDS) supplemented with 50mM DTT at 90°C for 2min. Proteolysis was achieved with three successive proteinase K treatments, performed by adding 400 µl of 2.4mg/ml proteinase K (Invitrogen AM2542) in 2× proteinase K buffer and incubating at 50°C for 30min, then adding 1.5mg/ml proteinase K in 250µl H2O and incubating at 50°C for 30min, and repeating this last step once more (the final volume is approximately 10 times that of the typical gel piece, which is around 100µl in volume). The shredded gel was removed using filter tubes (Millipore UFC30GV0S). The RNA was purified by phenol/chloroform/IAA extraction and precipitated using isopropanol and 15µg GlycoBlue™ (Thermo Fisher Scientific/Ambion, AM9516), then redissolved in 9 µl of H2O. 2µl of 10× RNA ligase buffer containing ATP (provided with Rnl1 T4 RNA ligase, Thermo Fisher Scientific, EL0021), 6µl of 50% PEG-8000, and 1 µl of 10 µM fPARclip 5′ adapter (Table S6) were added to the RNA sample. The RNA was denatured at 90°C for 1min and immediately cooled on ice for 2min. 2µl of Rnl1 T4 RNA ligase (Thermo Fisher Scientific, EL0021) was added, and the reaction mixture was incubated at 37°C for 1h. The RNA was purified using Oligo Clean & Concentrator™ (Zymo Research, D4060) and eluted with 11.5µl of H2O. cDNA was synthesized using fPARclip RT primer (Table S6) and SuperScript™ IV Reverse Transcriptase (Thermo Fisher Scientific, 18090010). The reaction mix was prepared according to the manufacturer's instructions. Briefly, 50pmol of fPAR-CLIP RT primer (Table S6) and 1µl of 10mM dNTP mix (each) were added to 11µl of adapter-ligated RNA sample, heated at 65°C for 5 min, and chilled on ice. 4µl of 5× SSIV Buffer, 1µl of 100mM DTT, 1µl of RNase inhibitor, and 1µl of SuperScript™ IV Reverse Transcriptase (200 U/µl) were added, and the reaction mix was incubated at 56°C for 30min. The reaction was inactivated by heating at 80°C for 10min and 60µl H2O was added to the reverse transcription reaction. Low-cycle PCR was performed using Platinum™ Taq DNA Polymerase (Thermo Fisher Scientific, 10966034), 5′ PCR primer short (Table S6), and fPARclip RT primer according to the manufacturer's instructions (10 µl of cDNA in 400 µl total, across 8 PCR tubes). The PCR programme was as follows: 94°C for 2min; 6 cycles of 94°C for 30s, 60°C for 30s, 72°C for 15s; 72°C for 1min. The amplified DNA was purified and concentrated using the DNA Clean & Concentrator™ kit (Zymo Research, D4013). Size selection of the amplified DNA was performed using the LightBench instrument (Yourgene Health) with a 3.0% agarose in a 12- channel in-channel filter size selection cassette (Yourgene health，CG-10600-13-300), where fragments between 78 and 100bp were extracted. The size selected sample was used as a template for a 1× 100 µl PCR reaction with 50 pmol Truseq PCR primers (Illumina) with the following PCR program: 94°C for 2min; 18 cycles of 94°C for 30s, 60°C for 30s, 72°C for 15s; 72°C for 1min. The fPAR-CLIP libraries were purified using DNA Clean & Concentrator™ kit (Zymo Research, D4013). Size selection of fPAR-CLIP sequencing libraries was performed using the LightBench instrument (Yourgene health). Briefly, libraries were run through a 3.0% agarose in a 12- channel in-channel filter size selection cassette (Yourgene health，CG-10600-13-300). Fragments between 145 and 175bp were extracted, corresponding to an expected fPAR-CLIP insert length of approximately 20 to 35 bp. The quality of the final library was assessed using the TapeStation system (Agilent). Sequencing was performed on an Illumina NovaSeq 6000 platform.