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| Status |
Public on Sep 03, 2019 |
| Title |
O_BRD4_1 |
| Sample type |
SRA |
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| Source name |
Human primary keratinocytes
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Human primary keratinocytes
input: BRD4_pooledInput
barcode: AGCCCTAA
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| Growth protocol |
All human cells were purchased from approved commercial suppliers who has obtained the necessary patient consent for skin donation for research purposes. The cells were obtained and handled in compliance with the Swiss Human Research Act. Normal human neonatal epidermal keratinocyte progenitor cells (nHPEKs, Lot #EB1110044 (pooled)) and adult normal human epidermal keratinocyte progenitor cells (aHPEKs Lots #ES1303277, and #MC1511045 (both abdominal)) were purchased from CELLnTEC (Bern, Switzerland) at Passage 2 (P2). HPEKs were expanded until Passage 4 (P4) and pellets of 1x106 cells were cryopreserved in liquid nitrogen for later use. HPEKs were cultured from P4 by seeding at an initial density of 5.3x104 cells/cm² using CnT-Prime media (CELLnTEC, #CnT-PR). When cells reached 70-85% confluency, they were detached from the cell culture dish using Accutase Cell Detachment Solution (CELLnTEC). For serial passaging experiments, aHPEKs were grown and passaged about 13 times. aHPEKs were collected for various assays and were considered as “HSCP-HKs” when fast-dividing (between Passages 7-9) whereas aHPEKs that were slow-dividing (Passages 15-17) were considered as “LSCP-HKs”.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For H3K27ac: Approximately 4x106 cells were cross-linked with 10% formaldehyde at room temperature for 15 min. After quenching with 125mM glycine for 6min, cells were washed in ice-cold PBS (plus Protease Inhibitor (PI) Cocktail tablet (Sigma) and 100mM PMSF) and collected by scraping in Nuclei EZ lysis buffer (Sigma, plus PI and 100mM PMSF). Nuclei were collected by centrifugation (500xg for 5min at 4°C). In order to increase nuclei collection, pellets were further re-suspended in a Hypotonic Buffer (10mM HEPES, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, pH8.0) for 10min in ice followed by NP40 (0.5% v/v) and 20sec vortex at maximum speed. Nuclei were collected by centrifugation (14.000rpm, 4°C, 30sec), washed with Wash Buffer C (with PI) (truChIP Chromatin Shearing Kit, Covaris, Brighton, UK) and re-suspended in Shearing buffer D3 (plus PI and 100mM PMSF) (truChIP Chromatin Shearing Kit, Covaris). Nuclei were vortexed for 3 rounds of 10sec and left 15min in ice before freezing in -80°C for later use. After thawing, 130μl nuclei suspensions were transferred into microTUBE (Covaris) and sonicated using a Covaris E220 (10min: duty cycle 2%, Intensity 5, Cycles per Burst 200). Samples were collected into 1.5ml tubes, centrifuged at 10000xg for 10min at 4°C and supernatants transferred into 2ml tubes. DNA concentrations were calculated after DNA isolation using Qiaquick PCR (Qiagen) using Qubit Nucleic Acid Quantification (Invitrogen). For ChIP a modified protocol of the Magna ChIP A/G (Merck #17-10085) protocol was used. In brief 5μg of mice anti-H3K27ac Ab or IgG were coupled with Protein A/G beads at 4°C for 4h. Coupled Ab-beads were mixed with 5μg of samples and incubated overnight at 4°C with rotation. Ab-beads were washed as per manufacturer’s instructions and protein/DNA complexes eluted in Elution buffer (50mM NaCl and 1%SDS in dH2O) for 4h at 65°C. Reverse cross-linking was performed by adding RNAse for 30min at 37°C and 5mM EDTA, 20mM Tris pH7.5 and 100μg/ml Proteinase for another 1h at 45°C. Supernatants were separated and DNA extracted using the MiniElute PCR purification kit (Qiagen).For BRD4: Frozen cells (9x106) were thawed and fixed with 1% formaldehyde for 15min and quenched with 0.125M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4μg of antibody against BRD4. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina’s NextSeq 500 (75nt reads, single end).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-seq for BRD4 in LSCP-HKs
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| Data processing |
All paired-end ChIP-seq datasets were aligned using Bowtie2 (version 2.2.8) to build version NCBI37/HG19.
Alignments were performed using all default parameters. We used the MACS version 1.4.2 (Model based analysis of ChIP-seq) (Zhang et al., 2008) peak finding algorithm to identify regions of ChIP-seq enrichment over background. A p-value threshold of enrichment of 1e-9 was used for all datasets.
Genome_build: hg19
Supplementary_files_format_and_content: Processed ChIP-Seq data files are in wiggle format from MACS output
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| Submission date |
Aug 10, 2019 |
| Last update date |
Sep 05, 2019 |
| Contact name |
Charles Yang Lin |
| E-mail(s) |
charles.y.lin@bcm.edu
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| Phone |
6172764723
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| Organization name |
Baylor College of Medicine
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| Department |
Molecular and Human Genetics
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| Street address |
1 Baylor Plaza
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| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE135676 |
IRF2 is a master regulator of human keratinocyte stem cell fate [ChIP-seq] |
| GSE135680 |
IRF2 is a master regulator of human keratinocyte stem cell fate |
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| Relations |
| BioSample |
SAMN12548602 |
| SRA |
SRX6692539 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4025774_O_BRD4_1.wig.gz |
107.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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