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| Status |
Public on Aug 08, 2013 |
| Title |
human_PolII_shGFP_ChIP_1 |
| Sample type |
SRA |
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| Source name |
HCT116 cell culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
antibody: Pol II N-20 (Santa Cruz catalog #sc-899)
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| Treatment protocol |
HCT116 cells were crosslinked with 1% formaldehyde for 10 min at room temperature with rotation and then crosslinking was quenched by the addition of glycine. Fixed chromatin was sonicated on a Misonix 3000 and used for immunoprecipitation with the indicated antibody. HCT116 cells were washed with cold phosphate-buffered saline (PBS) twice and lysed in RIPA buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol [DTT]) containing proteinase inhibitors (Sigma) for 30 min at 4°C. After centrifugation at 13,000 rpm for 30 min, the supernatant was incubated with the indicated antibodies and protein A/G PLUS agarose (Santa Cruz sc-2003) at 4 °C overnight with gentle rotation. The beads were spun down and washed three times with wash buffer (10 mM HEPES [pH7.4], 1 mM MgCl2, 300mM NaCl, 10mM KCl, 0.2%TritonX-100) before boiling in sodium dodecyl sulfate (SDS) loading buffer. HCT116 cells harvested following RNAi were infected with lentivirus harboring either Non-targeting shRNA or ICE1 shRNA in the presence of 8 µg/ml of polybrene (Sigma) for 24 hours (Target sequence for ICE1 sh1: 5’-ATAAGAGTCGTTTGCGAAATA-3’, Target sequence for ICE1 sh2: 5’-GCCTAATCAAGTATCAGTTAT-3’). The infected cells were selected with 2 µg/ml puromycin for extra 48 h before harvesting the cells.
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| Growth protocol |
For HCT116 cells, 5x10^7 cells were grown and used for each ChIP assay according to previously described protocols (Lee et al., 2006).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP libraries were prepared with the TruSeq DNA Sample Kit (Illumina #FC-121-2001) according to Illumina's instructions.
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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 following RNAi of GFP (non-targeting)
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| Data processing |
MACS peak detector v1.4.1 20110622; associated IP and input samples were configured with appropriately sized genome. Provided are bed file format for peaks meeting criteria: FDR < 5% and fold enrichment > 5.
Alignments were extended to a total fragment length of 150 bases in the orientation of the read alignment. UCSC bedGraph files were created at 25bp resolution and normalized to total alignable reads (reads-per-million).
Base-calling and quality filtering, default settings, Illumina Casava 1.8
Reads were aligned using Bowtie v0.12.7, allowing unique reads only and up to three mismatches of a 50bp seed length.
Peak detection was done using MACS, version 1.4.1 (20110622) using the indicated criteria.
RNA-seq alignments were done using TopHat v1.4.1 Differential expression analysis was done using DESeq corresponding to bioconductor version for R v2.11.1
Supplementary_files_format_and_content: http://genome.ucsc.edu/goldenPath/help/bedgraph.html
Genome_build: UCSC hg19 and UCSC dm3
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| Submission date |
Jun 13, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander (Garrett) Garruss |
| E-mail(s) |
asg@stowers.org
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| Organization name |
Stowers Institute for Medical Research
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| Lab |
Shilatifard
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| Street address |
1000 East 50th Street
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| City |
Kansas CIty |
| State/province |
Missouri |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE47938 |
Multiple roles for LEC in initiation and elongation phases of snRNA gene transcription |
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| Relations |
| BioSample |
SAMN02204102 |
| SRA |
SRX306182 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1162752_PolII_shGFP_150_rpm_25.bedGraph.gz |
387.0 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1162752_pol2_shGFP_peaks_pe-5.bed.gz |
330.9 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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