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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 09, 2017 |
| Title |
ChIP-seq-DGCR8-JQ1 |
| Sample type |
SRA |
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| Source name |
V6.5 embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
antibody: DGCR8
treatment: JQ1 (500 nM) 12 hr
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| Treatment protocol |
Cells were treated with DMSO or JQ1 (500 nM) for 12 hrs before ChIP-seq analysis.
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| Growth protocol |
V6.5 mESCs were cultured in mES media.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked for 10 min at room temperature by the addition of one-sixth of the volume of 6 % formaldehyde solution (diluted from methanol-free formaldehyde (Thermo Scientific)) to the growth media followed by 5 min quenching with 250 mM glycine. Cells were washed twice with cold PBS, then the supernatant was aspirated and the cell pellet was flash frozen in liquid nitrogen. Frozen crosslinked cells were stored at -80 ºC. Cell pellets were resuspended in ChIP Lysis buffer and ChIP Dilution buffer, treated with MNase for 30 min at -37 ºC followed by quenching with EDTA and EGTA, and further sonicated for 10 cycles at 30 s each with BioRupter. MNase-digested and sonicated lysates were cleared and incubated overnight at 4 ºC with 10 μg DGCR8 or Drosha antibody, and further incubated with pre-washed Dynabeads Protein G (Thermo Scientific) for 2 hrs. Beads were washed two times with sonication buffer, one time with sonication buffer with 500 mM NaCl, one time with LiCl wash buffer and one time with TE with 50 mM NaCl. After resuspension in elution buffer and reversal of crosslink at 65 ºC overnight, RNA and protein were digested using RNase A and Proteinase K, respectively, and DNA was purified with phenol chloroform extraction and ethanol precipitation.
After end-repair and A-tailing, ChIP DNA or whole cell extract DNA (50 ng) were ligated to diluted Illumina Adaptor Oligos and size-selected by two step AMPure bead selection (0.9x and 1.8x). After 16 cycle amplification with Phusion HF DNA polymerase, libraries were size-selected with 1.8x AMPure beads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
ChIP-seq samples were analyzed by Illumina HiSeq 2000 for 40 bases in single end read mode.
Reads were aligned to the mouse genome build mm9 using bowtie 1.0.1 (Langmead et al., 2009). The following parameters were used: -n 2, -e 70, -m 1, -k 1, and -l 40.
ChIP-seq peaks were called using MACS version 1.4.2. (model-based analysis of ChIP-seq) (Zhang et al., 2008) with a p value threshold of enrichment of 1 x 10–6.
Genome_build: mm9
Supplementary_files_format_and_content: Wig files were produced by MACS 1.4 with --space=100, and then converted to bw files in a rpm scale.
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| Submission date |
Nov 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hiroshi I. Suzuki |
| E-mail(s) |
hisuzuki-tky@umin.ac.jp
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| Organization name |
Nagoya University
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| Department |
DIvision of Molecular Oncology, Graduate School of Medicine
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| Street address |
65 Tsurumai-cho, Showa-ku
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| City |
Nagoya |
| ZIP/Postal code |
466-8550 |
| Country |
Japan |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE89810 |
Super-enhancer-mediated RNA processing revealed by integrative microRNA network analysis [X-ChIP-seq] |
| GSE89826 |
Super-enhancer-mediated RNA processing revealed by integrative microRNA network analysis |
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| Relations |
| BioSample |
SAMN06014898 |
| SRA |
SRX2345834 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2389983_ChIP_DGCR8_JQ1.bw |
401.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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