title: GSM2563629: RPF, 293T control-treated Rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Trizol, SDS/Proteinase K Clontech SMARTer RNA-seq or adapter ligation. Duplicate RNA-seq libraries for wild-type or ATF4-null HEK-293T cells treated with Torin 1 or tunicamycin for 6 h were prepared using the Clontech SMARTer Stranded Total RNA kit according to the manufacturer’s instructions. For ribosome profiling experiments, including both ribosome footprint (RPF) and total (RNA) libraries, HEK-293T cells were seeded in 15 cm dishes at 10 million cells/plate, incubated overnight, and then treated with vehicle (DMSO) or 250 nM Torin1 for 24 h. Cells were washed 3x in 5 ml cold PBS- and then lysed in polysome lysis buffer (15 mM Hepes pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton-X100). For RNA libraries, lysates were adjusted to 0.5% SDS, digested with proteinase K, and then extracted with acid-phenol. RNA was then fragmented by alkaline hydrolysis. RNA fragments of 40-80 nt were gel purified and used to prepare libraries as described below. For RPF libraries, lysates were digested with RNAse If for 45 min at RT, and centrifuged through 5-50% sucrose gradients to isolate monosomes. RNA was extracted from fractions containing the monosome peak by proteinase K digest, followed by acid-phenol extraction. Ribosome footprints were then isolated by excising and extracting 28-32 nt fragments from 15% polyacrylamide gels. Samples were depleted of abundant rRNA contaminants by hybridization with rRNA-specific biotinylated oligonucleotides. From this step, RNA and RPF libraries were treated identically. Samples were dephosphorylated using PNK, followed by 3’ ligation using T4 RNA ligase 1 to the 3’ linker, and ligation products were size selected by PAGE. Fragments were next 5’ phosphorylated using PNK, immediately followed by ligation to the 5’ linker using T4 RNA ligase 1. RNA was precipitated and subjected to reverse transcription using Superscript III with RT primer, after which RNA was destroyed by alkaline hydrolysis. RT products were purified by PAGE, and then amplified using the Phusion DNA polymerase. All samples were sequenced on an Illumina Hiseq system. Two biological replicates were prepared for each library.
attributes.tag: GEO Accession
attributes.value: GSM2563629