GSM6432852: CRIC-seq IgG IP (+pCp) rep2; Homo sapiens; OTHER
Cells were lysed and sonicated. RNA bound by the target protein was pulled down by a specific antibody. The RNA-protein complex was treated with protease K, and the RNA was extracted with TRIzol LS. To remove the potential joining of DNA to RNA mediated by T4 RNA ligase, 15 μl of RNA was treated with 10 μl of 10 × RQ1 DNase I buffer, 3 μl of RNase Inhibitor and 5 μl of DNase I at 37 °C for 20 min. RNA was purified with ZYMO RESEARCH RNA Clean and Concentrator-5.To fragment RNA, 16 μl of total RNA was added with 4 μl of 5 × first-strand buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2), incubated at 94 °C for 5 min and immediately put on ice. To enrich pCp-biotin-labeled chimeric RNAs, 20 μl of MyOne Streptavidin C1 beads (Thermo Fisher, 65002) were first washed twice with solution A (0.1M NaOH, 0.05M NaCl) and once with solution B (0.1M NaCl) and then blocked with yeast RNA (Roche, 10109223001). The blocked C1 beads, 30 μl of nuclease-free water, and 50 μl of 2 × TWB buffer were applied to the fragmented RNA. The mixture was incubated by rotating at RT for 30 min, after which the beads were washed four times with 1 × TWB buffer. To elute RNA from C1 beads, the beads were resuspended in 100 μl of PK buffer (100 mM NaCl, 10 mM Tris-HCl pH 7.0, 1 mM EDTA, 0.5% SDS) and incubated in Thermomixer at 95 °C for 10 min at 1000 rpm. Then the sample was placed on a DynaMag-2 magnet stand (Invitrogen, 12321D) for 1 min, and the aqueous phase was transferred to a new Eppendorf tube. Repeat the elution process once, and rinse the beads with 100 μl PK buffer. Finally, RNA was extracted from the 300 μl eluted sample with phenol chloroform 5:1 solution and precipitated overnight at a final concentration of 300 mM NaCl, with 3 volumes of ethanol and 1 μl of glycoblue at -20 °C. RNA was pelleted at 13,000 rpm, washed twice with 75% ethanol and dissolved in 10 μl of nuclease-free water.Strand-specific library construction for paired-end high throughput sequencing was performed as previously described (Cai et al., 2020). Briefly, first-strand cDNA was synthesized with random hexamer primer, and second-strand cDNA was synthesized by introducing dUTP in the dATP, dTTP, dCTP, and dGTP mix to achieve strand-specificity. Then the cDNA was performed end-repair, dA-tailing, and adaptor ligation, followed by PCR amplification.