GSM4145181: HeLa cells, WT (sg-SON) 0 hour rep 2; Homo sapiens; RNA-Seq
Total RNA was isolated by TRIzol reagent (Invitrogen). PolyA-RNA was isolated by two rounds of ployA selection with Oligo d(T)25 Magnetic Beads (NEB, S1419S) from total RNA and followed by additional TURBO DNase treatment. mRNA were purified by RNA Clean & Concentrator Kits and then fragmented to about 100 nt using RNA Fragmentation Reagents (Invitrogen, AM8740). The fragmented mRNA were purified by using RNA Clean & Concentrator Kits. For m6A-seq, while 100 ng mRNA was saved as input sample, the rest mRNA was used for m6A-immunoprecipitation. Briefly, nearly 5 ug mRNA was incubated with m6A antibody (Synaptic Systems, 202 003) and diluted into 500 uL IP buffer (150 mM NaCl, 0.1% Igepal CA-630, 10 mM Tris, pH 7.4, 100 U RNase inhibitor). The mixture was rotated at 4°C for 2 hours, following Dynabeads® Protein A (Invitrogen, 10002D) was added into the solution and rotated for another 2 hours at 4 °C. After four times washing by IP buffer, the m6A IP portion was eluted twice by 100 uL m6A-elute buffer (IP buffer, 6.7mM m6A, 30 U RNase inhibitor) with incubating and shaking at 4°C for 1 hour. Finally, m6A IP mRNA was recovered by RNA Clean & Concentrator Kits and the RNA concentration was measured with Qubit® RNA HS Assay Kit (Invitrogen, Q32855). Sequencing libraries of input and ip samples were prepared using NEBNext Ultra II RNA Library Prep Kit.
GEO Accession
GSM4145181