title: GSM6102498: Spinal cord, spared nerve injury, 14 day, rep3; Mus musculus; RIP-Seq
library_construction_protocol: The spinal cord of mice at 3rd, 7th, 14th day after spared nerve injury and sham group was isolated for RIP-Seq and RNA-Seq.Poly (A) RNA is purified from 50μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using 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.    Then 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).    Then the IP RNA was reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat.    1896649, USA), which were 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）.    Single- or dual-index adapters are 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 are amplified with PCR. The average insert size for the final cDNA library was 300±50 bp. Then we performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.