GSM8682866: Riboseq of KPC cells with Cis treatment for 21h,rep1; Mus musculus; OTHER
Cells were washed twice with cold PBS. 500 μl lysis buffer (8 mM HEPES, pH 7.4, 80 mM KCl, 4 mM MgCl2, 2% Triton x-100, and 200 μg/ml cycloheximide) was then added, and the cells were quickly scraped and collected into RNase-free tubes. The sample were centrifuged at 12,000 rpm for 12 minutes at 4°C, and the supernatant was sent to Novogene (China) for RNA-seq library construction. Approximately 100 μl of supernatant were treated with RNase I (Thermo Fisher Scientific) for 3 hours, followed by RNA extraction using TRIzol™ LS. The isolated RNA underwent denaturation at 65°C for 20 minutes and was then electrophoresed on a 15% TBE-Urea gel. The segment around 25-35 nucleotides was excised and eluted overnight in RNA elution buffer at 4°C. This was followed by precipitation of the RNA with an equivalent volume of isopropanol. The NEBNext Small RNA Library Prep Set for Illumina (E7330L, NEB, USA) was used for footprint library construction. The obtained DNA was then sent to Novogene for sequencing.