GSM5513431: sars2_IGF2BP1_rep2; Homo sapiens; RIP-Seq
The crosslinked cells were resuspended using 1 mL lysis buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS and 0.5% sodium deoxycholate) supplemented with protease inhibitor. Cells were lysed on ice for 30min. To fragment the RNA, 10 μL of RNase I dilution (1:1000 in lysis buffer) and 2 μL Turbo DNase were added, then the cell lysates were incubated for 3 min at 37 °C with shaking at 1100 rpm. The digested lysate was then immediately transferred to ice for 3 min, followed by centrifugation of lysates at 18,000 RCF for 10 min at 4 °C. IGF2BP1 antibody (10 μg, MBL, RN007P) or HSP90AB1 antibody (10 μg, Proteintech, 11405-1-AP) was conjugated to 100 μL protein A/G beads (Pierce, 88802). Rabbit IgG (10 μg) was also conjugated to protein A/G beads used for protein pull-down control. The conjugated beads were added to lysates and rotated for 1h at 4 °C. Beads were washed twice with lysis buffer and four times with wash buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.2% Tween-20); all washes lasted 5min with rotation at 4 °C. Co-purified RNA was dephosphorylated using T4 polynucleotide kinase (NEB, M0201L) following the manufacturer's instructions. The beads were washed twice with PNK wash buffer, following once with high-salt wash buffer (50 mM Tris-HCl, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% Igepal CA-630, 0.1% SDS and 0.5% sodium deoxycholate). Discard the high-salt wash buffer, beads were resuspended using PK buffer (100 mM Tris-HCl, pH 7.4, 50 mM NaCl, 10 mM EDTA) supplemented with 10 μL of proteinase K (20 μg/μL stock) then incubated at 37 °C for 20 min with shaking at 1100 rpm. TRIzol LS reagent (800 μL, Invitrogen, 10296028) was added to the tubes for RNA extraction as the manufacturer's instructions. The sequencing library of IGF2BP1 or HSP90AB1 uvCLAP was prepared as the protocol described in the SMARTer smRNA-Seq Kit for Illumina User Manual.