GSM4793041: batch1_RNASeq_ko; Mus musculus; RNA-Seq
Cells were put on ice, washed with ice-cold PBS supplemented with 100 ug/ml cycloheximide, lyzed in (20 mM Tris-HCl, pH 7.5 250 mM NaCl 1,5 mM MgCl2, 1mM DTT, 0,5% Triton X-100, 100 μg/ml cycloheximide and 20 U/ml TURBO DNAse). For total poly(A)-plus RNA sequencing, the lysates were deproteinized using Trizol-LS (Invitrogen) extraction, RNA was precipitated, purified via Oligotex resin (QIAGEN), and fragmented via mild alkaline hydrolysis. For ribosome footprints sequencing, the lysates were treated with RNase I (Ambion), the 80S monosomes were purified via 10-60% sucrose density gradient centrifugation, and deproteinized using acid phenol extraction. Both RNA_seq and ribo_seq RNA fragments were further purified via 15% PAGE and the RNA from 28 to 34 nt long was excised and extracted from gel slices. RNA was dephosphorylated using T4 PNK, then preadenylated Universal miRNA cloning linker (NEB) was ligated to RNA fragments using truncated T4 RNA ligase 2 (NEB), excised from 15% PAG, and reverse transcribed with SuperScript III revertase (Invitrogen). The DNA was excised from 7.5% PAG and circularized using CircLigase II (Illumina). The resulting libraries were depleted of rRNA-derived products, amplified with NEB TrueSeq adapters and sequenced.