GSM8366447: KO1_1 Selenium Supplementation; Homo sapiens; OTHER
cells were snap-frozen and  lysed in buffer containing 5 mM Mg2+ and 1% TritonX-100 with 100 μg/mL cycloheximide and tigecycline The Ribo-seq libraries were generated as previously described with following modifications (McGlincy et al.).  Cells were snap-frozen and allowed to thaw on ice, then lysed in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 μg/mL cycloheximide, 100 μg/mL tigecycline, 15 U/mL DNaseI (ThermoFisher, 89836)].  RNA was quantified using Qubit Broad range RNA kit (ThermoFisher, Q10211).  Ribosome footprint was generated using 15 U RNase I (Biosearch Technologies, N6901K) to 300 ug RNA in 200 μL reaction volume at room temperature for 45 min.  The ribosome was collected by ultracentrifugation and ribosome footprint was purified using TRIzol (ThermoFisher, 15596018).  RNA was then dephosphorylated and subjected to size selection from 17-40 nt range from 15% polyacrylamide TBE-Urea gel.  Ribosome footprint was extracted from the gel in the extraction buffer [50 mM Tris-HCl (pH 7.0), 300 mM NaCl, 1 mM EDTA, 0.25 % SDS] and subjected to rRNA depletion using Human/Mouse/Rat riboPOOL kit (Galen Molecular, dp-K024-000050) by following the manufacture's instruction.  rRNA-depleted ribosome footprint was precipitated in 80% ethanol at -20 °C overnight. rRNA-depleted ribosome footprint was then ligated to a pre-adenylated linker (rApp-WWAGATCGGAAGAGCACACGTC-3ddC).  The footprint was reverse transcribed by SuperScript III reverse transcriptase (ThermoFisher, 18080044) with a 5' phosphorylated primer that contains eight-nucleotide unique molecular identifiers and two spacer residues (Phos-NNNNNNNNAGATCGGAAGAGCGTCGTGTA-iSp18-AA-iSp18-TAGACGTGTGCTC).  cDNA was run on 10% non-denaturing gel to remove the non-ligated linker and the reverse transcription primer.  cDNA was the circularized using CircLigase I in the presence of 1 mM ATP.  The library was PCR amplified using KAPA HiFi HotStart ReadyMix (FisherScientific, 50-196-5217) and NEBNext® Multiplex Oligos for Illumina® Dual Index Primers Set 1 (NEB, E7600S).  The library was purified twice using 1x AMPure XP Bead (BeckmanCoulter, A63881).  The library was sequenced for single-end 100 cycles on Illumina NextSeq 2000.