title: GSM8459196: HEK293T, 1% Triton, Rep2, Ribo-Seq; Homo sapiens; OTHER
library_name: GSM8459196
library_construction_protocol: Cells were lysed with ice-cold polysome lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM MgCl2, in nuclease-free water, supplemented with 1 mM DTT, 0.1 mg/ml CHX, 25 U/ml Turbo DNase, and either 1%, 0.5%, or 0.1% Triton X-100) in triplicate. Lysates with centrifugation were clarified at 4°C for 5 mins at 10,000 x g. For Ribo-Seq, lysates of 15µg total RNA were treated with 35U RNase I (Biosearch Technologies) at 23°C for 1 hour at 400 rpm. Following T4 PNK end repair, ribosome-protected fragments were size-selected between 28-30nt via 15% TBE-urea polyacrylamide gel electrophoresis. Contaminating rRNA fragments were hybridised to a custom pool of 5'-biotinylated oligos with subsequent removal of these duplexes via affinity to streptavidin beads. A pre-adenylated linker bearing a unique molecular identifiers (UMI) was ligated to footprints. For RNA-seq, total RNA was extracted from lysates of 5µg RNA using TRIzol Reagent (Invitrogen) according to manufacturer's instructions. For Ribo-Seq, cDNA synthesis was carried out using the SMARTer smRNA-Seq Kit for Illumina (Takara Bio) according to manufacturer's instructions, excluding polyadenylation and using a custom reverse transcription primer instead of the 3' smRNA dT Primer provided, then amplified by 12-14 rounds of PCR. For RNA-seq, library construction was carried out with rRNA depletion using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions. Ribo-Seq, RNA-seq