title: GSM3537584: Young_HS1_RNA_Seq; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-Seq libraries: RNA was isolated from collected samples using the TRIZOL reagent (Thermo) following treatment with Turbo DNase and phenol chloroform precipitation. RNA-seq: After quality control, the samples were subjected to Illumina kit (TruSeq RNA) for library preparation. Ribosome footprint profiling libraries:  Ribosome footprinting was mostly performed as described in (Shalgi et al. 2013) with some modifications. Following heat shock, cells were lysed on ice for 10 minutes using the Lysis Buffer (see Cell culture and stress). Then the samples were treated with 20μl Turbo DNase for 5 minutes at 250C while shaking. Nuclei were removed with 10min spin at max speed, at 40C and supernatant was taken. 8 µl NEB RNase If for 2ml lysate at an OD260 of ~10 were added for 55min at 250C, shaking. Ribosomes were pelleted using a sucrose cushion (10ml RP lysis buffer + 12.5 ml 20 mM HEPES pH 7, 100 mM KCl, 5 mM MgCl2, 0.5 M Sucrose), by ultracentrifugation at 60K RPM for 1 hour and 50 minutes, at 40C. Ribosome pellets were subsequently resuspended in 0.5ml 8M guanidinium-HCl, and RNA was precipitated in acid phenol chloroform.
identifiers.namespace: GEO
identifiers.id: GSM3537584