title: GSM5653558: ASCC3 KD HEK293T #2 RNA-seq replicate 2; Homo sapiens; RNA-Seq
library_construction_protocol: Ribo-seq protocol: HEK293T cells were seeded at a density of 2.0 × 10^6 per 10-cm dish and the following analyses were performed 48 h after siRNA transfection. 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 CHX (100 μg/ml). 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 CHX (100 μg/ml)], 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. TCP-seq and Sel-TCP-seq protocol: Cells were seeded at a density of 8.0 × 10^6 cells per 15 cm dish, with 8 to 18 dishes per assay, and cultured for 2 days. Then, the subconfluent cells were treated for 10 min at room temperature with phosphate-buffered saline (PBS) containing 0.25% formalin, after which glycine was added to a final concentration of 125 mM and the cells were collected. The cells were washed three times with ice-cold PBS, lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, cOmplete EDTA-free protease inhibitor (Roche)] supplemented with cycloheximide (CHX) (100 μg/ml), 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 carefully, assayed for RNA concentration with a Qubit RNA BR assay kit, incubated with RNase I (20 U per 10 μg of RNA, Thermo Fisher Scientific) at 25°C for 45 min, and then placed on ice before the addition of 200 U SUPERase•In RNase inhibitor (Invitrogen). The samples were loaded on a 5% to 30% sucrose gradient buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, CHX (100 μg/ml), SUPERase•In (10 U/ml)] and centrifuged at 38,200 rpm (180,000 × g) for 4 h at 4°C in a Himac P40ST 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 FLAG 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 aliquots of the small ribosomal subunits (SSU) or large ribosomal subunits (LSU) fractions were stored for input or TCP-seq, and the remaining aliquots were incubated with the beads for 90 min at 4°C, and the resulting immunoprecipitates were washed four times with wash buffer [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1 mM DTT, 0.1% Triton X-100]. For Sel-TCP-seq analysis, 250 μl of decrosslinking buffer [1% SDS, 10 mM EDTA, 10 mM Tris-HCl (pH 7.5), 10 mM glycine], 750 μl of ISOGEN LS, and 200 μl of chloroform were added to the beads, and incubated for 45 min at 65°C, and RNA was then purified by precipitation with isopropanol. Ribo-seq protocol: 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). TCP-seq and Sel-TCP-seq protocol: The 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 subjected to dephosphorylation, DNA linker ligation, and rRNA depletion with RiboZero Gold as described for the Ribo-seq protocol. Reverse transcription 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 in a PCR reaction 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.