title: GSM6033156: OE_SLAM_GFP_3hr_B; Homo sapiens; OTHER
library_construction_protocol: For RNA-seq, cells were harvested with Tri-Reagent (Sigma-Aldrich), total RNA was extracted, and poly-A selection was performed using Dynabeads mRNA DIRECT Purification Kit (Invitrogen). For ribosome profiling, medium was aspirated from dishes, which were immediately placed on ice and rinsed with 10 ml ice-cold PBS supplemented with drugs used in pretreatment of the cells. PBS was aspirated and 800 μl ice-cold lysis buffer (20 mM Tris, pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1 mM dithiothreito, 0.5% Triton X-100 and 24 U / ml Turbo DNase (Ambion, AM2239), along with any drugs used for sample treatment) was dripped onto dishes. Cells were scraped and the lysate was removed and incubated 10 min on ice. The lysate was then clarified by centrifugation for 10 min at 20,000 × g, 4°C and ∼1.1 ml supernatant was recovered. RNA-seq: mRNA sample was subjected to DNaseI treatment and 3' dephosphorylation using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and T4 PNK (NEB) followed by 3' adaptor ligation using T4 ligase (NEB). The ligated products used for reverse transcription with SSIII (Invitrogen) for first strand cDNA synthesis. The cDNA products were 3' ligated with a second adaptor using T4 ligase and amplified for 8 cycles in a PCR for final library products of 200-300bp.