alias: GSM4395535
title: GSM4395535: TSB183_BJ_GRO_Senescent; Homo sapiens; OTHER
library_construction_protocol: GRO-seq was performed as described in Li et al., 2013(Li et al., 2013).  In brief, approximately 10 million BJ fibroblasts (either senescent or quiescent) were used per experimental condition.  Cells were incubated for 5 minutes on ice in swelling buffer (10mM Tris-Cl pH7.5, 2mM MgCl2, 3mM CaCl2), then lysed in lysis buffer (swelling buffer with 0.5% IGEPAL and 10% glycerol), and finally re-suspended in 100ul freezing buffer (50mM Tris-Cl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA).  The run-on reaction was then performed by mixing the re-suspended nuclei with an equal volume of run-on reaction buffer (10mM Tris-Cl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of Superase-In, 1% sarkosyl, 500?M ATP, GTP, and Br-UTP, 2?M CTP) and incubating 5 min at 30°C.  TRIzol LS reagent (Invitrogen) was then used, as per manufacturer's instructions, to extract nuclear run-on RNA (NRO-RNA). NRO-RNA was then hydrolyzed to ~300bp fragments via 35 minute incubation on ice in 0.2M NaOH, after which it was treated with DNase I and Antarctic phosphatase.  Following this, Br-UTP labeled NRO-RNA was purified using anti-BrdU agarose beads (Santa Cruz Biotech) incubated rotating at 4°C for 3h in binding buffer (0.5×SSPE, 1mM EDTA, 0.05% tween).  NRO-RNA then underwent end-repair using T4 PNK (NEB), after which cDNA synthesis was performed.      RNA fragments underwent a poly-A tairing reaction via incubation with poly-A polymerase (NEB) for 30 min at 37°C, after which reverse transcription was performed using superscript III (Invitrogen) with oNTI223 primer (Table 3).  cDNA products were then run on a 10% polyacrylamide TBE-urea gel, after which products between 100-500bp were cut and recovered by gel extraction.  Afterwards, first strand cDNA was circularized via CircLigase (Epicentre), and then re-linearized by treatment with Ape1 (NEB).  The re-linearized cDNA was then size selected on a TBE gel and fragments between ~120-320 bp were recovered via gel extraction.  This cDNA was then amplified via PCR using Phusion High-Fidelity enzyme (NEB) with oNTI200 ID1-6 (each sample is amplified with a different ID# primer to impart a distinct barcode) and oNTI201 (Table 3) for deep sequencing using the HiSeq 2500 system with single read runs of 50 cycles.
identifiers.namespace: GEO
identifiers.id: GSM4395535
attributes.tag: GEO Accession
attributes.value: GSM4395535