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| Status |
Public on Jul 24, 2016 |
| Title |
HP1BP3, a chromatin retention factor for co-transcriptional microRNA processing (ChIP-Seq) |
| Organism |
Homo sapiens |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Identification and characterization of HP1BP3 (a human histone H1 homologue) as a novel chromatin retention factor essential for the co-transcriptional processing of pri-miRNA.
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| Overall design |
We generated 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.
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| Contributor(s) |
Liu H, Kollipara RK, Kittler R, Liu Q |
| Citation(s) |
27425409 |
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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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| Platforms (1) |
| GPL11154 |
Illumina HiSeq 2000 (Homo sapiens) |
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| Samples (6)
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| This SubSeries is part of SuperSeries: |
| GSE77856 |
HP1BP3, a chromatin retention factor for co-transcriptional microRNA processing |
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| Relations |
| BioProject |
PRJNA311743 |
| SRA |
SRP069982 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE77854_DROSHA_ChIP.bed.gz |
96.2 Kb |
(ftp)(http) |
BED |
| GSE77854_H1_5_ChIP.bed.gz |
68.4 Kb |
(ftp)(http) |
BED |
| GSE77854_HP1BP3_ChIP.bed.gz |
150.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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