GSM7336625: m6Apool2_IP_ESC_CAST-HOUS_rep1; Mus musculus; RIP-Seq
Yeast total RNA samples were prepared by adjusting previously published protocols. After fast freezing the yeast cells in liquid nitrogen, yeast cells were resuspended in equal amounts of phenol:Chloroform:Isoamyl alcohol (Sigma Aldrich), buffer AE (50mM sodium acetate, 10mM EDTA 1% SDS), and glass beads in Eppendorf tube. The tube was vortexed for 15 minutes in a bullet blender, heated to 65 °C for 30 minutes, followed by another round of 5 minutes vortex and 30 minutes heating to 65 °C. Samples were placed on ice for 5 minutes and centrifuged for 10 minutes (12000g, 4 °C). The supernatant was isolated, re-extracted with phenol:Chloroform:Isoamyl alcohol, and precipitated with sodium acetate and isopropanol. For all types of mammalian cell lines, total RNA was extracted with BIO TRI RNA reagent (Bio-lab). Enrichment for mRNA was done by two rounds of poly-A selection using Oligo(dT) beads (Dynabeads mRNA DIRECT) in both total RNAs from yeast and mammals. m6A-seq2: Fragmentation, 3' adapter ligation, pooled(m6A-IP, cDNA synthesis, second adapter ligation and enrichment) m6A-seq2