GSM4368209
GSM4368209: input siCtrl rep2; Homo sapiens; OTHER
For m6A-IP,total RNA was purified by TRIzol reagent (Invitrogen), following the manufacture's instruction. mRNA was extracted from the total RNA by using Dynabeads mRNA Purification Kit (Ambion) following the instruction. The rRNA was further removed by RiboMinus Eukaryote Kit (Invitrogen). mRNA was fragmented by sonication. The m6A-immunoprecipitation was performed using NEB EpiMark N6-Methyladenosine Enrichment Kit (E1610S). For CLIP, FTO or TRMT10A overexpression cells were UV crosslinked three times in a Stratagene Stratalinker 2400 UV Crosslinker. After irradiation, cells were harvested by cell lifter and pelleted by centrifugation at 500g for 4 min at 4 ℃. The pellet was then resuspended in lysis buffer (1 x PBS supplemented with 0.1% SDS, 1% Nonidet P-40, 0.5% Sodium Deoxycholate, Protease Inhibitor Cocktail) and kept on ice for 15 min. T1 digestion was carried out under 2 U/μl for 15 min at 22 °C. 200 μl anti-Flag beads (Millipore, M8823) were subsequently added to the supernatant and kept at 4 °C for overnight with rotation. The beads were then washed for 3 times with 1 ml of high salt buffer (5 x PBS supplemented with 0.1% SDS, 1% Nonidet P-40 and 0.5% Sodium Deoxycholate) and another 3 times with 1 ml of wash buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl2 and 0.2% Tween-20). The RNAs were then isolated by 200 μl of elution buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, and 1% SDS) containing 2 mg/ml proteinase K under incubation at 37 °C for 40 min. The beads were removed and RNAs were recovered by RNA Clean & Concentrator (Zymo Research). m6A profiling libraries were constructed by TruSeq stranded mRNA samples preparation kit from Illumina. CLIP-seq libraries were constructed by NEBNext® Small RNA Library Prep Set for Illumina (NEB7330).
GEO
GSM4368209
GEO Accession
GSM4368209