Name: GSM7111084
Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cells were quickly washed with ice cold PBS supplemented with 100 µg/ml cycloheximide (CHX) and snap-frozen in liquid nitrogen. For libraries prepared with cycloheximide in the lysis buffer, plates were thawed on ice and cells were scraped off the plate in 400 µl polysome lysis buffer (20 mM Tris pH=7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton-X100, 1 mM DTT, 100 µg/ml CHX, 25 U/ml Turbo DNase), 0.1% NP-40, 10 µg/ml aprotinin, 20 µM leupeptin, 2.5 µM pepstatin A, 0.5 mM AEBSF and 1x Phosphatase Inhibitor Cocktail. Samples were vortexed vigorously, triturated through a 26G gauge needle, and spun down for 7 minutes at 16,000xg/ 4°C. Supernatant was transferred to a new tube. 20 µg RNA in 200 µl polysome lysis buffer were digested with 50 U RNase I for 45 minutes at 2,000 rpm/22°C. For libraries prepared with cycloheximide/tigecycline (CHX/TIG) in the lysis buffer, plates were thawed and cells from a 10-cm dish were lysed in 15 ml polysome lysis buffer supplemented with 0.1% NP-40 and 100 µg/ml tigecycline (TIG). After incubation on ice for 5 minutes, extracts were pre-cleared by centrifugation for 5 minutes at 3,000 g/ 4°C. Ribosomes were pelleted through 3 ml of a sucrose cushion (1 M sucrose, 20 mM Tris pH=8.0, 140 mM KCl, 5 mM MgCl2, 1 mM DTT) by spinning the layered solutions in the Type 70 Ti rotor for 120 minutes at 50,000 rpm/ 4°C. Ribosome pellets were rinsed once, dissolved in 200 µl drug-free polysome lysis buffer, and incubated with 200 U (hiPSC) or 300 U (NPC) RNase I for 45 minutes at 2,000 rpm/22°C. Ribosome footprint libraries were prepared essentially as described (McGlincy and Ingolia, 2017; Wu 2019) with minor modifications. RNase I digestion was stopped by addition of 100 U Superase In and extracts were loaded on a sucrose cushion. The pellet was dissolved in 400 µl LiDS/LET lysis buffer and RNA was extracted with the acid phenol protocol. Fragments in the range of 19 to 32 nucleotides were isolated by gel size selection, with T4 PNK and ligated to pre-adenylated adapters containing 5 random nucleotides at their 5' ends (McGlinzy 2017) with T4 RNA Ligase 2, truncated KQ. Adapter-ligated RNA was subjected to rRNA depletion using the Ribo-Seq riboPOOL h/m/r depletion kit (siTOOLs) for CHX-only samples, and legacy RiboZero Gold kit (Illumina) for CHX+TIG samples The rRNA-depleted footprints were reverse-transcribed with Protoscript II and cDNA was circularized with recombinant TS2126 RNA ligase 1 (commercially available as CircLigase). Libraries were constructed from circularized cDNA with KAPA HiFi DNA Polymerase and single-end sequencing was performed on a NextSeq 550 platform (Illumina).