GSM5291915: eIF2D KO HEK293T #1 arsenite RNA-seq replicate 2; Homo sapiens; RNA-Seq
The medium was aspirated, and the cells were immediately placed on ice, washed once with ice-cold phosphate-buffered saline (PBS), and lysed with lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and EDTA-free protease inhibitor] supplemented with either CHX (100 μg/ml) for Ribo-seq or 50 μM LTM for GTI-seq. The lysates were then incubated with TURBO DNase on ice for 10 min and centrifuged at 15,000 rpm for 10 min at 4°C, and the resulting supernatants were collected carefully, assayed for RNA concentration with a Qubit RNA BR assay kit, incubated with RNase I (20 U per 10 μg of RNA, Epicentre) for 45 min at 25°C, and placed on ice before the addition of SUPERase•In RNase inhibitor (Invitrogen). The samples were then loaded on a 1 M sucrose cushion buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, SUPERase•In (10 U/ml), 1 mM DTT, and either CHX (100 μg/ml) for Ribo-seq or 50 μM LTM for GTI-seq], and the gradients were centrifuged at 100,000 rpm for 1 h at 4°C in a Beckman TLA110 rotor. The resulting pellets were suspended in ribosome splitting buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA, SUPERase•In (20 U/ml), 1 mM DTT, 1% Triton X-100] and subjected to purification with the use of an Amicon Ultra filtration device (100-kDa cutoff, Millipore) to deplete rRNAs. The purified RNA was subjected to selection on the basis of a size range of 17 to 34 nt by electrophoresis through a 15% polyacrylamide and Tris-borate-EDTA (TBE)–urea gel (SuperSep RNA, Fujifilm). The footprint fragments were treated with T4 PNK to repair the 2x-3x cyclic phosphates, a DNA linker including barcode sequences (NI-810 to NI-817) was ligated with the use of T4 RNA Ligase 2, truncated K227Q (New England Biolabs), and the resulting products were purified on a 15% polyacrylamide and TBE-urea gel. Ribosomal RNAs were further depleted with the use of RiboZero Gold (Illumina). Reverse transcription was performed with the NI-802 primer, and the resulting products were purified on a 15% polyacrylamide and TBE-urea gel. The purified cDNAs were circularized with circLigase II (Lucigen), and index sequences were then added by amplification in a PCR reaction with the common primer (NI-798) and primers including index sequences (NI-799 and NI-822 to NI-826). Products of the desired size were purified on a 15% polyacrylamide nondenaturing gel (SuperSep DNA, Fujifilm), and the libraries were sequenced with a NovaSeq 6000 system (Illumina).