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| Status |
Public on Mar 19, 2019 |
| Title |
H3K9me3_ChIPSeq_RPE Exp2 |
| Sample type |
SRA |
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| Source name |
RPE
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| Organism |
Mus musculus |
| Characteristics |
tissue: retinal pigment epithelium (RPE)
strain: C57BL/6
chip antibody: H3K9me3 (Diagenode Cat# C15410193)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Freshly isolated RPE were cross-linked in 1% formaldehyde with 1X PBS. To stop the cross linking reaction, glycine was added to a final concentration of 0.125M. The chromatin was prepared using the True MicroChIP Kit (Diagenode Cat# C01010130). Chromatin was sheared using the Bioruptor® Pico sonication device (Diagenode Cat# B01060001). ChIP was performed using IP-Star® Compact Automated System (Diagenode Cat# B03000002) following the protocol of the aforementioned kit. Chromatin corresponding to 14,000-28,000 cells was immunoprecipitated using the antibodies.
Libraries were prepared from input and ChIP’d DNA (500 pg) using the MicroPlex Library Preparation Kit v2 (12 indices) (Diagenode Cat# C05010013). Library amplification is assessed using the High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). Libraries were then purified using Agencourt® AMPure® XP (Beckman Coulter) and quantified using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854). Finally their fragment size was analyzed by High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Advanced Analytical Technologies, Inc.). When the proportion of fragments >500bp was too high, libraries were subjected to a double size selection using Agencourt® AMPure® XP (Beckman Coulter). Libraries were pooled and sequenced on an Illumina HiSeq 4000 with single-end reads of 50bp lengths, running HiSeq Control Software HD version 3.4.0.38.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Quality control of sequencing reads was performed using FastQC.
Reads were then aligned to the reference genome (mm10) obtained from the UCSC genome browser using BWA software v.0.7.5a.
Samples were filtered for regions blacklisted by the ENCODE project. Subsequently samples were deduplicated using SAMtools version 1.3.1.
Alignment coordinates were converted to BED format using BEDTools v.2.17 and peak calling was performed using SICER with customized parameters for each histone mark.
Genome_build: mm10
Supplementary_files_format_and_content: BED files were generated using SICER with customized parameters for each histone mark. The first column is the name of the chromosome; second - the starting position of the peak; third - the ending position of the peak; fourth - the name of the peak; fifth - the p-value of the peak
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| Submission date |
Sep 27, 2018 |
| Last update date |
Mar 19, 2019 |
| Contact name |
Dmitry Ivanov |
| E-mail(s) |
divanov@med.miami.edu
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| Phone |
305-482-4230
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| Organization name |
University of Miami Miller School of Medicine
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| Department |
Ophthalmology
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| Street address |
1638 NW 10th Avenue
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| City |
Miami |
| State/province |
FL |
| ZIP/Postal code |
33136 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE120590 |
Genome-wide maps of chromatin state in retinal pigment epithelium (RPE) of adult mice |
| GSE120592 |
Gene expression, chromatin state and DNA methylome in retinal pigment epithelium (RPE) |
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| Relations |
| BioSample |
SAMN10139602 |
| SRA |
SRX4746220 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3405389_RPE_Exp2_H3K9me3_peaks.bed.gz |
156.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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