GSM4824272: healthy1_input; Homo sapiens; RIP-Seq
Total RNA was purified with TRIzol reagents from human heart samples. Polyadenylated RNA was then isolated from total RNA with two rounds of polyA tail purification using Dynabeads® mRNA DIRECT™ kit (Thermo Scientific). rRNA was further removed from polyadenylated RNA using RiboMinus Eukaryote kit (Thermo Scientific). 50 ng polyadenylated RNA after rRNA depletion was digested by nuclease P1 (Sigma, N8630), respectively, in 20 μl of buffer containing 25 mM NaCl and 2.5 mM ZnCl2 for 1 h at 42 °C. 1 ug polyadenylated RNA was adjusted to 10 ng/μl in 100 μl and fragmented using a BioRuptor ultrasonicator (Diagenode) with 30 s on/off for 30 cycles. After fragmentation, 5% of the fragmented RNA was saved as input. m6A marked fragments were enriched using EpiMark N6-Methyladenosine Enrichment Kit (NEB) following the manufacturer's protocols and collected as IP. Both input and IP samples were subjected to library preparation using TruSeq Stranded mRNA Library Prep Kit (Illumina) following the manufacturer's protocols Both input and IP samples were subjected to library preparation using TruSeq Stranded mRNA Library Prep Kit (Illumina) following the manufacturer's protocols.