GSM4273149: m6A_Col0_rep2; Arabidopsis thaliana; OTHER
Total RNA was first extracted  by Trizol reagent, The polyA RNA was then purified with oligo d(T)25 magnetic beads For m6A-Seq, polyA RNA was fragmented by NEBNext Magnesium RNA Fragmentation Module. A portion of fragmented RNA was reserved as m6A input, others were incubated with anti-m6A antibody and eluted as the m6A IP. Both m6A input and IP samples were prepared for next-generation sequencing library by standard protocol of NEBNext Ultra II RNA Library Prep Kit for Illumina; For A-Seq2, poly(A) RNA was fragmented by alkaline hydrolysis and performed with 5' ends phosphorylation and DNase treatment. 3' ends were blocked by cordycepin triphosphate and 5' endwere ligated with 3' adaptor.  Reverse transcription (Superscript IV, Invitrogen) was carried out using Bio­dU­dT25, then the USER enzyme and RNase H (NEB) was added to get the first strand cDNA. After 5' adaptor ligation, cDNA was amplified and sequenced with Illumina HiSeq X Ten; For FA-CLIP, formaldehyde cross-linked samples were harvested and lysated, and the lysates were treated with Turbo DNase and RNase T1 and incubated with pre-washed Anti-Flag M2 magnetic beads. After washed by low-salt wash buffer, beads was suspended into low-slat wash buffer and digested with RNaseT1. Beads was incubated with T4 PNK and ATP orderly to end repair. Using protease K digestion and phenol/chloroform extraction, the recovered RNA were prepared for next-generation sequencing followed by standard protocol of NEB Next Multiplex Small RNA Library Prep Kit for illumina; For RNA-Seq, RNA libraries were prepared for next-generation sequencing using standard protocol of NEBNext Ultra II RNA Library Prep Kit for Illumina
GEO Accession
GSM4273149