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| Status |
Public on Aug 13, 2015 |
| Title |
N20_shSCR_MCF7_ChIPSeq |
| Sample type |
SRA |
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| Source name |
MCF7
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| Organism |
Homo sapiens |
| Characteristics |
cell type: breast cancer cell line
cell line: MCF7
shRNA: Scramble shRNA
chip antibody: Pol II (Santa Cruz, sc-899)
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| 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
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| Extracted molecule |
genomic DNA |
| 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-seq of Total Pol II
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| 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
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| Submission date |
Jun 30, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
ash@northwestern.edu
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| Organization name |
Northwestern University Feinberg School of Medicine
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| Department |
Department of Biochemistry and Molecular Genetics
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| Lab |
Shilatifard Lab
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| Street address |
320 E Superior St
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE70408 |
PAF1, a molecular regulator of promoter-proximal pausing by RNA Polymerase II |
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| Relations |
| BioSample |
SAMN03835635 |
| SRA |
SRX1078883 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1727113_N20_shSCR_MCF7_ChIPSeq.bw |
139.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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