GSM8730090: B16F10 shRNA OSGEP#2 replicat1; Mus musculus; RNA-Seq
B16F10 cells depleted or not for OSGEP were grown to confluence in a 10 cm dish and treated with 10 µg/mL cycloheximide for 5 minutes at 37ºC to arrest translation, followed by cell lysis and RNA digestion. Active ribosomes were then pulled down using a puromycin analog (RiboLace probe) and magnetic beads, with two pulldowns performed for each sample. RNA was subsequently extracted with the acid phenol-chloroform method, followed by size selection of 25-35 bp fragments in a 15% TBE-urea PAGE gel using Immagina's PAGExt gel extraction system. RPFs were quantified with the Qubit™ microRNA Assay Kit before proceeding with the library preparation RPF library preparation with LACEseq involved sequential 5' phosphorylation, ligation, and circularization steps, with small RNA purification after each step using the RNA Clean & Concentrator™-5 kit from Zymo Research. Reverse transcription and an initial PCR amplification were then performed, followed by cleanup of the PCR product using AMPure XP beads. A second PCR amplification was carried out to incorporate unique dual indexes using Immagina's iUDI Plate. Final cleanup of the indexed library was done using the NucleoSpin Gel and PCR Clean-Up kit followed by size selection in a 10% TBE PAGE gel with PAGExt gel extraction Library quality and size was confirmed on a Bioanalyzer, and sequencing was conducted on a NovaSeq (Illumina) at the NGS platform of the GIGA Institute, with 70 million reads per sample.