GSM5461441: ON LPS cKO Input rep2 m6A-seq; Mus musculus; RIP-Seq
Total RNA was harvested from activated cells using Trizol. Enrichment for PolyA RNA was done twice using mRNA mRNA direct kit and checked using TapeStation (Agilent). PolyA-selected RNA was barcoded and immunoprecipitated in two rounds using anti-m6A polyclonal antibody bound to magnetic protein-G beads and protein-A beads Libraries were prepared as previously described (Garcia-Campos et al. Cell, 2019). Briefly, eluted RNA was reverse transcribed and an adapter was added using Superscript III reverse transcriptase (Thermo Fisher, 18080093). A second adaptor was added to the cDNA by ligation with T4 RNA Ligase 1 (NEB, M0437M). Following clean-up with MyOne Silane beads, the cDNA library was amplified in a PCR using KAPA HiFi HotStart ReadyMix (KAPA Biosystems KK2601).Libraries were sequenced on an Illumina NextSeq 500 machine.