title: GSM8491808: 32D cell KO2 RPF rep2; Mus musculus; OTHER
library_construction_protocol: 500 µl lysate was prepared as described. 100 µl lysate was used to prepare total RNA library, meanwhile, ribosome footprinting and subsequent library preparation of ribosome protected RNA fragments (RPFs) was performed with 400 µl lysate. Add 10 U RNase I to 400 µl lysate and incubate for 45 min at room temperature with gentle mixing. Then the nuclease digestion was stopped by adding 1 µl RNase inhibitor. RPFs were purified with MicroSpinS-400 columns (GE Healthcare Life Sciences) followed by size selection, which was conducted using 15% TBE-urea gel. rRNAs were depleted with the NEBNext rRNA depletion kit (New England BioLabs). Following end repair and 3'adaptor ligation, RNAs were reverse transcribed using SuperScript III (Thermo Fisher, USA). cDNA libraries were prepared as described (Nat Protoc. 2012 Jul 26;7(8):1534-50) and sequenced with Illumina NovaSeq 6000 with strand-specific paired-end 150 bp read length.