title: GSM5600256: m1Aseq-Alkb-WT1 CD4 T cell-6h; Mus musculus; OTHER
library_construction_protocol: RNAseq: total RNA was extracted based on the manufacturer's protocol by using TRIzol reagent (Invitrogen). Riboseq: we performed the ribosome profiling by strictly following the manual for the Illumina TruSeq Ribo Profile (Mammalian) Kit. m1Aseq: 200 ng small RNA fraction (<200 nt; mainly composed of mature tRNA) was purified from total RNA using MEGAclear Kit (Thermo Fisher Scientific). RNAseq:RNA libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB) based on the manufacturer's instructions. Riboseq: libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (no. E7300S, E7300L). m1Aseq: Purified small RNA was deacylated by incubating in 0.1 M Tris-HCl, pH 9 at 37°C for 45 min. Half of the deacylated small RNA fraction was subjected to demethylation treatment by using AlkB in vivo reaction. RNA samples were dephosphorylated with PNK (NEB) and ligated to 3' RNA linker using T4 RNA ligase2, truncated KQ (NEB). Reverse transcription was carried using different reverse transcriptase for different samples. 5' adaptor was ligated to cDNA and then amplified by PCR. PCR products were purified by 8% TBE gel. RNAseq, Riboseq, m1Aseq.