GSM8774641: siControl HEK293T Ribo-seq replicate #2; Homo sapiens; OTHER
RNA-seq extract protocol Total RNA was extracted from cell lysates with the use of a PureLink RNA Mini Kit (Thermo Fisher Scientific), and the quality of the purified RNA was assessed with a 2100 Bioanalyzer (Agilent). Ribo-seq extract protocol cells were seeded at a density of 3.0 × 106 per 10-cm dish and cultured overnight to ensure subconfluence. The culture medium was removed, and the cells were immediately placed on ice, washed once with ice-cold PBS, and lysed with a lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-100, and EDTA-free protease inhibitor cocktail] supplemented with cycloheximide (100 μg/ml). The lysates were incubated on ice for 10 min with TURBO DNase (Thermo Fisher Scientific) and then centrifuged at 20,380 × g for 10 min at 4°C, and the resulting supernatants were collected, assayed for RNA concentration with a Qubit RNA BR Assay Kit (Thermo Fisher Scientific), 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 [containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, SUPERase•In (10 U/ml), 1 mM dithiothreitol, and cycloheximide (100 μg/ml)], and the gradients were centrifuged at 100,000 rpm (417,200 × g) 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 dithiothreitol, and 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). Sel-TCP-seq extract protocol Cells were seeded at a density of 1.0 × 107 cells per 15-cm dish, with 8 dishes per assay, and were cultured overnight. The subconfluent cells were treated for 15 min at room temperature with crosslinking buffer [PBS containing 0.025% formaldehyde, 0.5 mM dithiobis(succinimidyl propionate) (DSP), 10 mM MgCl2, and 100 μg/ml cycloheximide], after which glycine was added to a final concentration of 125 mM and the cells were collected. The cells were washed twice with ice-cold PBS, lysed in a lysis buffer [50 mM HEPES-KOH (pH 7.5), 200 mM KCl, 10 mM MgCl2, 1% NP40, 100 μg/ml cycloheximide and cOmplete EDTA-free protease inhibitor cocktail (Roche)], and incubated for 10 min on ice. The lysates were centrifuged at 20,380 × g for 10 min at 4°C, and the resulting supernatants were collected, quantified for RNA concentration with a use of Nanodrop spectrophotometer (Thermo Fisher Scientific), incubated with RNase I (100 U per 120 μg of RNA, Thermo Fisher Scientific) at 5 min on ice. The samples were immediately loaded on a 15% to 50% sucrose gradient [containing 20 mM Tris-HCl (pH 7.5), 160 mM KCl, and 10 mM MgCl2] and centrifuged at 35,000 rpm (210,000 × g) for 5 h at 4°C in a SW41Ti rotor. The gradient was fractionated with the use of a TRIAX gradient-profiling system and AC-5700P microcollector. Polysome profiles were determined by measurement of absorbance at 260 nm. Dynabeads Protein G were conjugated with antibodies to V5 by incubation overnight at 4°C in lysis buffer supplemented with 0.5% bovine serum albumin, and they were washed three times with lysis buffer before use. Small portions of the 40S fractions were stored for TCP-seq, and the remaining samples were incubated with the beads for 90 min at 4°C for Sel-TCP-seq. The resulting immunoprecipitates were washed four times with a lysis buffer. 250 μl of de-crosslinking buffer [1% SDS, 10 mM EDTA, 10 mM Tris-HCl (pH 7.5), 5 mM DTT and 10 mM glycine], 750 μl of ISOGEN LS (Nippon Gene), and 200 μl of chloroform were added to the beads (Sel-TCP-seq) or 40S fractions (TCP-seq), which were then incubated for 45 min at 65°C before purification of RNA by precipitation with isopropanol. RNA-seq library construction protocol For RNA-seq, after mRNA selection with the use of a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), libraries were prepared with the use of a NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs). For 3′ mRNA-seq, libraries were prepared from 1µg of the purified total RNA with the use of QuantSeq 3' mRNA-Seq V2 Library Prep Kit FWD with UDI 12nt Set A1 for Illumina platform (Cat no.191.96, LEXOGEN). 16 cycles of PCR were used to amplify the library. The libraries were sequenced with a NovaSeq 6000 system (Novogene Japan). Ribo-seq library construction protocol The footprint fragments were treated with T4 polynucleotide kinase (New England Biolabs) to repair the 2'-3' 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 (RT) 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 with the polymerase chain reaction (PCR) as performed 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). Sel-TCP-seq library construction protcol Purified RNA was selected according to size in the range of 17 to 100 nt by electrophoresis in a 15% polyacrylamide and TBE-urea gel, and was then subjected to dephosphorylation, DNA linker ligation, and rRNA depletion with RiboZero Gold as described for the Ribo-seq protocol. RT was performed with the use of the SI-019 primer, and the resulting products were purified on a 15% polyacrylamide and TBE-urea gel. The second linker (SI-018) was ligated by incubation overnight at 25°C with T4 RNA Ligase 1 (ssRNA Ligase), High Concentration (New England Biolabs). The products were then amplified by PCR with the same primers as used for the Ribo-seq protocol, and those of the desired size were purified by electrophoresis through a 15% polyacrylamide nondenaturing gel. The libraries were sequenced with a NovaSeq 6000 system.