GSM7566891: RPF M395 RES shVARS-1_3; Homo sapiens; OTHER
Frozen lysate of patient derived melanoma cells has been delivered to the OHMX facility. Total RNA from 2 µg of the lysate was purified with the RNA Clean and Concentrator-5 kit (Zymo Research). 10 µg of lysate was digested with RNase I (Thermofisher) at 21°C for 45 min. The reaction was stopped with the addition of Superase In RNase Inhibitor (Thermofisher). The digested lysate was purified on a MicroSpin S-400 HR Sephacryl column (GE Healthcare). The flow through was collected and purified with the RNA Clean and Concentrator-5 (ZymoResearch). The footprints were visualized on the Agilent Bioanalyzer with the RNA 6000 Nano kit (Agilent). Ribosome-protected fragments (RPFs) of 28-30 nt were selected by loading the footprints on a Novex TBE-Urea gel (15%) (Thermofisher) and performing gel electrophoresis RPFs were 3' dephosphorylated with T4 PNK (New England Biolabs)  and were subsequently purified using the Oligo Clean and Concentrator kit (ZymoResearch). The 3' adapter was ligated to the RPFs and the ligated fragments were purified following the protocol of the Small RNA Library prep kit (Lexogen). After the 5' phosphorylation of the RPFs and their purification with the Oligo Clean and Concentrator kit, 5' adapter ligation took place based on the Small RNA Library prep kit. First strand cDNA synthesis was performed according to the Small RNA Library prep kit manual. Ribosomal RNA (rRNA) was depleted with the addition of LNA probes from the QIAseq FastSelect rRNA HMR kit (Qiagen) during the cDNA synthesis reaction. During PCR, a unique index primer (Lexogen) was added to one end of the fragment.The PCR amplification protocol from the Small RNA Library prep kit was followed.