title: Ribo-seq (HHT) of D458 medulloblastoma cell line
design: Matched homoharringtonine ribosome profiling data was generated for D283, DAOY, ONS76, R262,UW228, D384, D458, D425, Med411, SUMB002, Med2112, CHLA-01-MEDR, and D341.  Ribosome profiling was performed as detailed previously (CITE HOFMAN). In brief, cells were grown to 60-70% confluence.  Cells were then pre-treated with 5ug/mL homoharringtonine for 10 minutes.  Cells were then harvested, washed once in 1x cold PBS, and collected by centrifugation.  Cells were lysed in a buffer of 20mM Tris HCl, 150mM NaCl, 5mM MgCl2, 1mM dithiotrietol, 0.05% NP-40 and 25U/mL Turbo-DNase I (Invitrogen).  2ug/mL of cyclohexamide was additionally added to the lysis buffer.  Cleared lysates were quantified and 2.5U/ug of RNAse I was added for 45 minutes at room temperature for digestion.  The reaction was quenched with an equal volume of 1U/uL Superase RNase Inhibitor (Ambion).  Ribosome protected fragments (RPFs) were isolated using ultracentrifugation at 55,000RPM at 4C for 2 hours.  RPF RNA was purified with a Zymo Direct-Zol kit (Zymo) and rRNA was depleted using the siTOOLS human riboPOOL kit (siTOOLS Biotech, Germany).  Denatured RPFs were resolved on a 15% TBE-Urea gel (200V, 65 minutes) and the RPF band at 26-32 nt was excised and RNA extracted.  After RNA precipitation, RNA was end-repaired with T4 PNK at 37C for 1 hour, cleaned up with RNA Clean and Concentrator kit (Zymo).  The 3 linker adapter was ligated (22C, 3hrs) using T4 RNA ligase I and R4 RNA ligase 2 deletion mutant.  Linker reactions were removed with Rec J exonuclease (Lucigen) and 5 deadenylase (NEB).  cDNA was synthesized with EpiScript RT enzyme (Lucigen, 50C for 30 minutes).  After clean-up with Exonuclease I and RNAse I and hybridase (Lucigen), cDNA was resolved on a 10% TBE-Urea gel (175V for 1 hour), and 75nt bands were excised and DNA was precipitated.  Precipitated cDNA was circularized with CircLigase (Lucigen, 60C for 3hrs).  Circular cDNA was amplified using 2x Phusion HiFi master mix (NEB) in a 11-14 cycle PCR reaction depending on cDNA yield.  PCR products were precipitated with ethanol and 5M NaCl, and resolved on a 8% TBE gel (100V, 90 minutes).  The 150nt band was excised, purified, quantified for DNA abundance using a DNA Qubit, and analyzed for library size using an Agilent TapeStation (Agilent).  Libraries were then sequenced on an Illumina NovaSeq to achieve 150  250 million reads per sample.