GSM5591842: HEK293T_CHX_rep1; Homo sapiens; OTHER
Ribosome profiling was carried out according to the previously described protocol (McGlincy, N. J.; Ingolia, N. T. Methods 2017, 126, 112–129.) with minor modificationsCells were lysed and the lysates were clarified by centrifugation for 10 min at 20,000g, 4 °C. For each condition, lysate containing 30 μg total RNA (determined by Qubit RNA BR Assay Kit, Thermo Fisher Scientific) was used for nuclease footprinting. The digestion was transferred to 8 x 34 mm thick wall polycarbonate ultracentrifuge tube (Beckman 343776) on top of 0.25 mL sucrose cushion. Ribosome recovery was carried out by centrifugation in a TLA 100.1 rotor at 100,000 rpm, 4 °C for 3 h 40 min. The RNA from the pelleted ribosomes was purified first using the Direct-zol RNA Miniprep kit (Zymo Research) and then by precipitating with isopropanol. Size selection by running a 15% polyacrylamide TBE-Urea gel for 65 min at 200 V in 1X TBE, staining the gel for 3 min with 1X SYBR Gold in TBE, and excising the 17 nt – 34 nt region. The size-selected RNA was extracted in the gel extraction buffer and purified by isopropanol precipitation. The extracted RNA was resuspended in 10 μL 10 mM Tris pH 8, and end repaired by T4 polynucleotide kinase (New England Biolabs, hereafter NEB). Linker ligation was performed using T4 RNA ligase 2 truncated K227Q (NEB) and linkers preadenylated using 5' DNA adenylatioin kit (NEB). NI-812 linker (barcode CGTAA) was used for footprints from CHX-treated cells and NI-810 (barcode ATCGT) was used for footprints from 8- treated cells. Depletion of the unligated linker from the ligation reaction was carried out by adding 1 μL yeast 5'-deadenylase 10 U/μL and 1 μL RecJ exonuclease 10 U/μL (epicenter definition) directly to the ligation reaction and incubating for 45 min at 30 °C. After pooling and purification of ligations, ribosomal RNA was depleted using RiboMinus™ Eukaryote System v2 (Thermo Fisher Scientific) according to the manufacturer's instruction. The resulting RNA was reverse transcribed using SuperScript III RT (Invitrogen) and the reverse transcription products were separated from the unextended primers and no-insert background products by running a 15% polyacrylamide TBE-Urea gel for 40 min at 180 V. The reverse transcription products were extracted from the excised bands in the DNA gel extraction buffer and were purified by isopropanol precipitation. The resulting cDNA was circularized by CircLigase II (EpiCentre) and was quantified by qPCR. Library construction was carried out by 9 cycles of PCR using Q5 Hot Start High-Fidelity DNA Polymerase (NEB). After purification of the DNA library, library length distribution was analyzed by TapeStation 2200 (Agilent) using High Sensitivity D1000 ScreenTape. The first technical replicate was sequenced for 51 cycles (paired end) on an Illumina MiSeq, and the second replicate was sequenced for 150 cycles (single read) on an Illumina NextSeq. Ribosome profiiling