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Sample GSM1727123 Query DataSets for GSM1727123
Status Public on Aug 13, 2015
Title nascent.RNA_shPAF1.1_HCT116_rep2
Sample type SRA
 
Source name HCT116
Organism Homo sapiens
Characteristics cell type: colorectal cancer cell line
cell line: HCT116
shRNA: PAF1 shRNA #1
Growth protocol HCT116 and MCF7 cells were grown in DMEM supplemented with 10% FBS. S2 cell were grown in Schneider's media with 10% FBS at room temperature
Extracted molecule nuclear RNA
Extraction protocol For ChIP-seq, HCT116 cells were crosslinked with 1% paraformaldehyde for 10 minutes at room temperature with gentle rotation, and then quenched by 0.125 M glycine solution. After washing, nuclei were sonicated on a Misonix Sonicator 3000 Ultrasonic Cell Disruptor, and the supernatant was used for immunoprecipitation with the indicated antibody. ChIP-sequencing libraries were prepared with Illumina’s Tru-seq DNA sample prep kit. For nascent RNA-seq, HCT116 cells was harvested and washed 3 times with cold PBS, then suspended in 10 ml Buffer A (10 mM HEPES at pH=7.9, 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 1× Complete protease inhibitors [Roche]). After incubating on ice for 15 minutes, the cells were homogenized in pre-cooled 15 ml Dounce tissue homogenizer for 15 times. Nuclei were then washed twice with Buffer B (10 mM HEPES at pH=7.9, 250 mM Sucrose, 1 mM DTT, 1× Complete protease inhibitors). The pellet was vigorously suspended with 1 ml NUN buffer (20 mM HEPES at pH=7.9, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% v/v Nonidet P40, 1 mM DTT, 20 U/ml SUPERase.In RNase Inhibitor [Ambion]) that was freshly prepared. The chromatin was then washed twice more with 5 ml NUN buffer each time. The supernatant was removed and RNA was purified. The RNA was subjected to polyA depletion with Oligo(dT) magnetic beads (Invitrogen) and DNase I treatment (NEB) for 20 minutes, and then was re-purified. For sequencing, 2 μg of resulting RNA was used for ribosomal RNA depletion with the RiboZero kit (Epicenter) and libraries were made with the TruSeq RNA sample Prep Kit (Illumina).
ChIP-sequencing libraries were prepared with Illumina’s Tru-seq DNA sample prep kit using standard protocols. Nascent and Total RNA-seq libraries were prepared with Illumina’s TruSeq RNA sample Prep Kit using standard protocols. The global nuclear run-on procedure and the preparation of Gro-seq libraries were previously described (Core et al., 2008; Gardini et al., 2014).
 
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina HiSeq 2000
 
Description Nascent RNA-seq
Data processing Base-calling and quality filtering, default settings, Illumina Casava 1.8 for reads generated with Illumina HiSeq 2000
Base-calling and quality filtering, default settings, bcl2fastq version 2 for reads generated with Illumina NextSeq 500
ChIP-seq: Reads were aligned using Bowtie v0.12.9, allowing unique reads only and up to two mismatches in the entire read length. Alignments were extended to a total fragment length of 150 bases toward the interior of the sequenced fragment. UCSC bigWig files were created with 1bp resolution coverage across the genome and normalized to total reads per million (r.p.m).
Nascent and Total RNA-seq: Reads were aligned to the human genome (UCSC hg19) and Ensembl 72 GTF human annotations using the Tophat aligner version 2.0.10, with the - g1 option. Alignments were not extended. UCSC bigWig files were created with 1bp resolution coverage across the genome and normalized to total reads per million (r.p.m).
Genome_build: UCSC hg19
Supplementary_files_format_and_content: http://genome.ucsc.edu/goldenPath/help/bigWig.html
 
Submission date Jun 30, 2015
Last update date May 15, 2019
Contact name Ali Shilatifard
E-mail(s) ash@northwestern.edu
Organization name Northwestern University Feinberg School of Medicine
Department Department of Biochemistry and Molecular Genetics
Lab Shilatifard Lab
Street address 320 E Superior St
City Chicago
State/province IL
ZIP/Postal code 60611
Country USA
 
Platform ID GPL11154
Series (1)
GSE70408 PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II
Relations
BioSample SAMN03835645
SRA SRX1078893

Supplementary file Size Download File type/resource
GSM1727123_nascent.RNA_shPAF1.1_HCT116_rep2.minus.bw 157.5 Mb (ftp)(http) BW
GSM1727123_nascent.RNA_shPAF1.1_HCT116_rep2.plus.bw 163.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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