title: GSM6463600: HeLa_polyA_WT_FTO-_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were cultured to 70-80% confluency, rinsed with 1× PBS (Gibco), and lysed by the direct addition of TRIzol reagent (Invitrogen). Total RNA was then collected following the manufacturer's protocol. Poly A+ RNA was extracted from purified total RNA using Dynabeads mRNA DIRECT Purification Kits (Invitrogen). Modification-free control RNAs were prepared from HeLa or mESC total RNA according to a previously published protocol (PMID: 34594034, 32518404). RNA was annealed to 2 pmol of RT primer (AGACGTGTGCTCTTCCGATCT) at 70ºC for 2 min, then reverse transcribed in 1× RT buffer (Thermo Scientific, EP0753, containing 50 mM Tris-HCl, pH 8.3; 75 mM KCl, 3 mM MgCl2, 10 mM DTT) with 5 mM of RNaseOUT (Invitrogen, 10777019) and 200 U of Maxima H- Reverse Transcriptase (Thermo Scientific, EP0753) at 50ºC for 1h. The cDNA was released by boiling the beads in 0.5% (v/v) SDS for 10 min. The eluate was purified by DNA Clean & Concentrator (Zymo) kits. Purified cDNA was ligated to cDNA Adapter (/5Phos/NNNNNN AGATCGGAAGAGCACACGTCTG/3SpC3/) using 30 U of T4 RNA ligase (NEB) at 25ºC overnight, following a previously published protocol (PMID: 35288668). The reaction was purified again by DNA Clean & Concentrator (Zymo) kits and then PCR amplified with NEBNext Ultra II Q5 Master Mix (NEB, M0544X) and NEBNext Unique Dual Index Primer for Illumina (NEB, E6440S) following the manufacturer's directions. Typically, 10-11 cycles of PCR were carried out to generate enough DNA. The resulting library was purified by AMPure XP beads (Beckman Coulter, A63882) following the manufacturer's directions and submitted for next-generation sequencing.