title: Illumina HiSeq 2000 paired end sequencing; Global Run-On sequencing (GRO-seq) of HeLa cells under CDK9 inhibitor tratment to study elongation checkpoints at both ends of RNA polymerase II-transcribed genes.
design: Global Run-On sequencing (GRO-seq) of HeLa cells under CDK9 inhibitor tratment to study elongation checkpoints at both ends of RNA polymerase II-transcribed genes.
library_construction_protocol: HeLa cells were grown in DMEM medium supplemented with 10% fetal calf serum, 100U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine at 37ºC and 5% CO2 HeLa cells were either untreated (control) or treated with 100 micromolar KM05283 (Maybridge) or 100 micromolar DRB (Sigma) for 30 minutes. 1.5 x 107 HeLa cells were harvested by centrifugation, washed with PBS, and resuspended in HLB (10 mM Tris pH 7.5, 10 mM NaCl, 2.5 mM MgCl2) + 0.5% NP-40 + 2 units (U)/ml SUPERase In (Ambion). After 5 min incubation on ice, nuclei were pelleted through a cushion of HLB + 0.5% NP-40 + 10% sucrose + 2U/ml SUPERase In. Nuclei were resuspended in an equal volume of transcription buffer (10 mM Tris pH 8, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 20U SUPERase In, 1% Sarkosyl, 500 uM ATP, GTP and Br-UTP, 2 uM CTP). The transcription reaction was carried out at 30°C for 5 min and terminated by centrifugation for 30 seconds (sec). Nuclei were resuspended in HSB (10 mM Tris pH 7.5, 0.5 M NaCl, 10 mM MgCl2) containing 10U of RNase-free DNase and incubated at 30°C for 20 min. Protein was digested by the addition of 200 μg/ml of proteinase K in buffer (20 mM Tris-HCl pH 7.4, 2% SDS, 10 mM EDTA) at 55°C for 1 hour (h). RNA were extracted twice with acid phenol:chloroform, once with chloroform and precipitated with 3 volumes of -20°C ethanol containing 30 mM NaCl and glycogen. The pellet was washed with 75% ethanol before resuspending in 20 l of DEPC-treated water. Base hydrolysis, immunoprecipitation of Br-U RNA and end-repair were performed as described by Core et. al. Science 322, 1845-8 (2008) The run-on library was prepared by the High-Throughput Genomics Group at The Wellcome Trust Centre for Human Genetics. Two untreated controls, KM-treated and DRB-treated samples were multiplexed together and sequenced on a single lane of Illumina HiSeq using a standard 51 nucleotides (nt) paired-end reads protocol.
attributes.tag: Experimental Factor: compound
attributes.value: DRB
attributes.tag: Experimental Factor: dose
attributes.value: 100 micromolar
attributes.tag: LIBRARY_STRAND
attributes.value: first strand