title: Ribo-seq (DMSO) of UW228 medulloblastoma cell line
design: Ribo-seq for cancer cell lines was performed based upon the protocol by McGlincy et al., Methods, 2017, with modifications as described below. Briefly, cells were grown to 60-70% confluence prior to collection. After collection, all cell pellets were washed once in 1x PBS, re-pelleted by centrifugation, and lysed in lysis buffer (20mM Tris HCl, 150mM NaCl, 5mM MgCl2, 1mM dithiotrietol, 0.05% NP-40, 25U/mL Turbo-DNase I (Invitrogen), 2ug/mL cycloheximide). After clearing the lysate and recovering the supernatant, RNA abundance was determined by measuring the A260. 2.5U/ug of RNase I was added to an appropriate volume of lysate and incubated at 22C for 45 minutes without shaking. The RNase I was then quenched with 1U/uL of Superase RNase Inhibitor (Ambion). RNA from ribosome protected fragments were recovered using a 1M sucrose cushion with ultracentrifugation (55,000 RPM, 4C, 2 hours), and rRNA was depleted using the siTOOLS human RiboPool kit according to manufacturers instructions (siTOOLS Biotech, Germany). Ribosome protected fragments were then denatured using a 1:1 mixture with 2x sample loading buffer (98% v/v formamide, 10mM EDTA, 300ug/mL bromophenol blue) at 95C for 3 minutes, and further purified using size selection from a 15% TBE-Urea gel (200V for 65 minutes). The 26 32 nucleotide band was cut from the gel, RNA extracted by freezing gel slices in 400uL RNA gel extraction buffer (300mM NaOAc, 1mM EDTA, 0,25% v/v SDS), and rotating at room temperature for 5-6 hours. RNA was precipitated with 500uL isopropanol and 2.0uL GlycoBlue at -20C overnight; pellets were washed once in chilled 70% ethanol, and subjected to end-repair with T4 PNK (Lucigen, 37C for 1 hr). End-repaired RNA was cleaned up with the RNA Clean and Concentrator kit (Zymo), ligated to a 3 linker (sequence below, 6.67% w/v PEG-8000, 6.67 mM dithiotrietol, 1x T4 RNL2 Truncation buffer, 6.67 U/uL R4 RNA ligase 2 Deletion mutant, 0.33 U/uL T4 RNA ligase I) for 3 hours at room temperature. Linker reactions were removed with 5 deadenylase (New England Biolabs) and Rec J Exonuclease (NEB), and cDNA was generated with EpiScript RT enzyme (Lucigen, 50C for 30 minutes) followed by reaction clean up with exonuclease I (Lucigen, 37C for 30 minutes), RNase I/Hybridase (Lucigen, 55C for 5 minutes) and the Oligo Clean and Concentrator Kit (Zymo). cDNA was mixed 1:1 with 2x sample loading buffer, boiled, and purified with a 10% TBE-Urea gel (70 minutes, 175V). The product between 70 90 nucleotides was excised from the gel, and DNA was extracted with 450uL DNA extraction buffer (300mM NaCl, 10mM Tris, 1mM EDTA, 0.02% SDS) with a flash-freeze on dry ice (30 minutes) and rotation at 22C for 6 hours. DNA was precipitated with 700uL isopropanol and 2uL GlycoBlue at -80C overnight followed by centrifugation at 14,500 RPM for 45 minutes at 4C. DNA pellets were washed once in 80% ethanol and pellets were air-dried and dissolved in 11uL of water, which was then circularized with the addition of 9uL of CircLigase I mix (1M betaine, 1x CircLigase Buffer (Lucigen), 2.5mM MnCl2, 50uM ATP, 5U/uL CircLigase I (Lucigen)) at 60C for 3 hours with heat inactivation at 80C for 10 minutes. Circularized cDNA was quantified using quantitative real-time PCR (10uL of 2x SYBR-Green mastermix (Thermo), 2uL of cDNA, 6uL water, 1uL forward and reverse primer each) for twenty cycles, using the following PCR primers (JRP_qPCR-ribo-F2 primer: CAGAGTTCTACAGTCCGACGAT; JRP_qPCR-ribo-R2 primer: AGACGTGTGCTCTTCCGATCT). Library PCR amplification was performed with 10uL of 2x Phusion HiFi master mix (New England Biolabs), 8uL of cDNA sample, 1uL of the forward library primer (AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGACG) and 1uL of the appropriate barcorded reverse primer (Table S4L). PCR reactions were run with the following cycle conditions: 98C for 1 minute, followed by 12-15 cycles of 94C for 16 seconds, 55C for 6 seconds, and 65C for 11 seconds, with a final extension of 65C for 1 minute. PCR products were mixed with 6x gel loading buffer and size-selected on a 8% TBE gel, 100V for 75 minutes. The product at ~150 bps was gel-excised, placed in 400uL of DNA extraction buffer, flash frozen on dry ice for 30 minutes, thawed at 22C for 6 hours on a rotating platform, and DNA was precipitated with 700uL of isopropanol with 2uL of GlycoBlue overnight at -80C. Samples were then centrifuged at 14,500 RPM for 45 minutes; DNA pellets were washed once in 80% ethanol, air-dried, and dissolved in 18uL of 5mM Tris. Samples were quantified by DNA Qubit (Thermofisher) and library size was confirmed using an Agilent Bioanalyzer HS DNA High Sensitivity Kit (Agilent). Libraries were sequenced at the Dana-Farber Molecular Biology Core Facility on an Illumina NovaSeq 6000.