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| Status |
Public on Jul 24, 2016 |
| Title |
DROSHA_ChIP |
| Sample type |
SRA |
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| Source name |
BAC transgenic Hela cell lines
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
antibody: Polyclonal (goat) against full length His-EGFP
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| Treatment protocol |
Not applicable
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| Growth protocol |
HeLa cells were cultured at 37 °C in 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin /streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
BAC transgenic cells at 80% confluency (~1x107) were cross-linked with 1% formaldehyde for 10 minutes at 37°C, and quenched with 125 mM glycine at room temperature for 5 minutes. The fixed cells were washed twice with cold PBS, scraped, and transferred into 1 ml PBS containing protease inhibitors (Roche). After centrifugation at 700 g for 4 minutes at 4°C, the cell pellets were resuspended in 100 μl ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.1] with protease inhibitors) and sonicated at 4°C with a Bioruptor (Diagenode) (30 seconds ON and 30 seconds OFF at highest power for 15 minutes). The sheared chromatin with a fragment length of ~200 – 600 bp) was centrifuged at 20,000 g for 15 minutes at 4°C). 100 μl of the supernatant was used for ChIP or as input. A 1:10 dilution of the solubilized chromatin in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl 16.7 mM Tris-HCl [pH 8.1]) was incubated at 4°C overnight with 6 μg/ml of a goat anti-GFP (raised against His-tagged full-length eGFP and affinity-purified with GST-tagged full-length eGFP). Immunoprecipitation was carried out by incubating with 40 μl pre-cleared Protein G Sepharose beads (Amersham Bioscience) for 1 hour at 4°C, followed by five washes for 10 minutes with 1ml of the following buffers: Buffer I: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl; Buffer II: 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl; Buffer III: 0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]; twice with TE buffer [pH 8.0]. Elution from the beads was performed twice with 100 μl ChIP elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature (RT) for 15 minutes. Protein-DNA complexes were de-crosslinked by heating at 65°C in 192 mM NaCl for 16 hours. DNA fragments were purified using QiaQuick PCR Purification kit (QIAGEN) and eluted into 30 μl H2O according to the manufacturer’s protocol after treatment with RNase A and Proteinase K.
barcoded libraries of DROSHA-GFP, HP1BP3-GFP and H1.5-GFP ChIP and input DNA were generated with the TruSeq® ChIP Sample Preparation Kit (Illumina)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
DROSHA_ChIP.bed
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| Data processing |
Basecalls were performed using CASAVA (v. 1.8.2)
ChIP-seq reads were aligned to hg19 geneome assembly using Bowtie (v.2.2.5)
Mapped reads were filtered to remove low quality, ambiguous and redundant reads
Peaks were called using the SICER (v. 1.1)
Genome_build: hg19
Supplementary_files_format_and_content: Processed data files contains DROSHA, HP1BP3 and H1.5 peak coordinates in BED format
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| Submission date |
Feb 11, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ralf Kittler |
| E-mail(s) |
Ralf.Kittler@UTSouthwestern.edu
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| Organization name |
University of Texas Southwestern Medical Center
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| Street address |
5323 Harry Hines Blvd
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| City |
Dallas |
| State/province |
Texas |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE77854 |
HP1BP3, a chromatin retention factor for co-transcriptional microRNA processing (ChIP-Seq) |
| GSE77856 |
HP1BP3, a chromatin retention factor for co-transcriptional microRNA processing |
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| Relations |
| SRA |
SRX1571793 |
| BioSample |
SAMN04501337 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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