RIP-seq_PNT_D3_STM_IP (CRX1630677)
Total RNA was subjected to fragmentation (~150 nt) using chemical hydrolysis, followed by end-repair with T4 Polynucleotide Kinase. Barcoded 3' adapters (adenylated, ligated via T4 RNA ligase 2) were appended, and excess adapters were removed by Lambda Exonuclease treatment. RNA was immunoprecipitated with m6A-specific antibody (Cell Signaling Technology, 56593) conjugated to Protein G beads. Enriched RNA fragments (IP) and input control RNA were ligated to 5' adapters using T4 RNA ligase . First-strand cDNA was synthesized with HiScript III reverse transcriptase (Vazyme), and libraries were amplified via PCR (KAPA HiFi HotStart Ready Mix) with adapter-specific primers.