GSM8919815: HepG2, caRNA, Nm-mut-seq, Treat, rep1; Homo sapiens; OTHER
For purification of chromatin-associated RNA, 10 million HepG2 cells were washed in PBS and harvested at 500g for 3 mins. Cells were lysed by 200 μL cold NP-40 lysis buffer (10 mM Tris pH 7.5, 0.15% NP-40, 150 mM NaCl) and suspended by flicking the tube without pipetting. After lysing on ice for 5 mins, The lysate was layered over 200 μL cold sucrose cushion (10 mM Tris pH 7.5, 0.15% NP-40, 150 mM NaCl, 24% sucrose) and centrifuged at 15,000g for 10 mins at 4°C. The pellet was wash with PBS and suspended by 100 μL cold glycerol buffer (20 mM Tris pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% Glycerol) by flicking the tube without pipetting, followed by mixing with 100 μL cold nuclei lysis buffer (10 mM HEPES pH 7.6, 7.5mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1M Urea, 1% NP40) and vortexing at highest speed for 6 times of 10 sec each. The lysate was incubated on ice for 2 mins followed by centrifugation at 15,000 g for 10 mins at 4°C. The pellet was further washed with PBS and digested with TURBO DNase followed by proteinase K. RNA was purified by TRIzol. rRNA was removed by RiboMinus™ Eukaryote System v2, and the residual RNA was cleaned up by RNA Clean & Concentrator™-5 (Zymo Research, R1014) with removal of short RNA < 200 nt. RNA was fragmented by RNA Fragmentation Reagent (Invitrogen, AM8740), end repaired and ligated with 3′ linker 5'rApp-NNNNNATCACGAGATCGGAAGAGCACACGTCT-3SpC3. The residual adapter was removed with 5'-deadenylase and RecJf, purified and ligated with 5′ SR adapter supplied in the NEBNext® Small RNA Library Prep Set. The ligated product was separated in halves and used to perform RT reaction with balanced (1 mM dNTP each, named Ctrl) or biased (2  μM dG/C/TTP and 1 mM dATP, named Treat), followed by RNaseH digetion and PCR amplification. Sequencing by Illumina NovaSeq X was conducted with single-end 100 bp.