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| Status |
Public on Jan 05, 2015 |
| Title |
Pol_II_shScr_starved_rep2_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Colon cancer cell line
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| Organism |
Homo sapiens |
| Characteristics |
strain: HCT116
cell type: human colon cancer cell
antibody: RNAPII rabbit polyclonal N20 (Santa Cruz, sc-899)
serum: starved
targeted by: scramble shRNA
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| Growth protocol |
HCT116 cells were grown in DMEM supplemented with 10% FBS.
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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 TRIzol reagent (Invitrogen) was added to the pellet for purification of RNA. 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-seq and Nascent-RNA-seq libraries were prepared for sequencing using standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Chromatin IP of Pol II in starved HCT116 cells targeted by scramble shRNA
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| Data processing |
Base-calling and quality filtering, default settings, Illumina Casava 1.8
Reads were aligned using Bowtie v0.12.9, allowing unique reads only and up to three mismatches of a 50bp seed length.
Nascent-seq alignments were done using TopHat v2.0.9 with bowtie1 v0.12.9, using -g 1 option and allowing up to two mismatches.
ChIP-seq: Alignments were extended to a total fragment length of 150 bases in the orientation of the read alignment. UCSC bigWig files were created at 1bp resolution and normalized to total alignable reads (reads-per-million).
Nascent-seq: Reads were aligned using Ensembl 67 GTF human annotations using TopHat v2.0.9 and Bowtie v0.12.9. Alignments were not extended. UCSC bigWig files were created at 1bp resolution and normalized to total alignable reads (reads-per-million).
Genome_build: UCSC hg19
Supplementary_files_format_and_content: http://genome.ucsc.edu/goldenPath/help/bigWig.html
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| Submission date |
Nov 14, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Joan Conaway |
| E-mail(s) |
[email protected]
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| Organization name |
Stowers Institute
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| Street address |
1000 E 50th ST
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| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE63314 |
Stably-paused genes revealed through inhibition of transcription initiation by the TFIIH inhibitor Triptolide |
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| Relations |
| BioSample |
SAMN03195899 |
| SRA |
SRX759965 |
| Named Annotation |
GSM1545669_Pol_II_shScr_starved_rep2_ChIPSeq.bw |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1545669_Pol_II_shScr_starved_rep2_ChIPSeq.bw |
111.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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