title: GSM7030303: SCC-9 cells, NC2_input; Homo sapiens; RIP-Seq
library_construction_protocol: total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and quantified. RNA integrity was evaluated by Bioanalyzer 2100 (Agilent, CA, USA). Poly (A) RNA was purified using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) and fragmentedinto about 100nt using Magnesium RNA Fragmentation Module (NEB, USA). The RNA fragments weredivided into two portions, one was kept as input and the otherwas used to enrich m6A-methylated RNA fragments by immunoprecipitation with m6A-specific antibody (Synaptic Systems, Germany). Poly (A) RNA was purified from 50μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) by two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 86℃ 7min. The cleaved RNA fragments were incubated for 2h at 4℃ with m6A-specific antibody (No. 202003, Synaptic Systems, Germany) in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which was next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA）. An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform(LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol MeRIP-seq