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| Status |
Public on Apr 01, 2021 |
| Title |
H3K9me3_ChIPseq_Tex191ESC_KO3Input |
| Sample type |
SRA |
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| Source name |
H3K9me3_ChIPseq_Tex191ESC_KOInput
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| Organism |
Mus musculus |
| Characteristics |
strain background: 129/Ola
cell type: Embryonic stem cells
passages: 12-20
genotype/variation: Tex19.1-/-
chip antibody: none (input)
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| Growth protocol |
Mouse embryonic stem cells derived from the parental E14Tg2a line were cultured in gelatinised flasks in Glasgow Modified Eagle’s Media (GMEM), 10% foetal calf serum, 1% non-essential amino acid, 1% sodium pyruvate, 1% penicillin-streptomycin, 1% L-glutamine, 0.001% β-mercaptoethanol, and 0.2% leukaemia inhibitory factor-conditioned media at 37°C in 5% CO2 and passaged using 0.05% trypsin, 0.02% EDTA in PBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryonic stem cells were detached from 70% confluent T75 flasks by scraping and resuspended in ice-cold PBS. Cross-linked ChIP was then performed essentially as previously described (Mortazavi et al., 2006, Genome Res. 16, 1208–1221) by fixing cells with formaldehyde, collecting nuclei from lysates, sonicating then isolating histone-DNA complexes with anti-histone H3K9me3 antibody (Abcam ab8898). Samples of input DNA were removed prior to the addition of anti-histone H3K9me3 antibodies.
Libraries were prepared using an NEBNext Ultra ChIPseq kit and sequenced using an Illumina HiSeq 4000 75PE sequencing system.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Input DNA
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| Data processing |
Sequences aligned to the mouse mm10 reference genome with Bowtie2 v2.2.6 using settings: -X 500 --sensitive.
The alignment maps were converted into tag directories using HOMER v4.8 makeTagDirectory using settings: -sspe -unique. Peaks were found from the 'IP' sample relative to its 'Input DNA' sample for each replicate using HOMER v4.8 findPeaks using settings: -style histone.
Peaks common to at least two replicates of the same genotype were identified and merged using BEDTools v2.25.0.
Read coverage for the peak intervals was determined using HOMER v4.8 annotatePeaks using settings: -size given.
Read counts were normalised to the number of mapped reads (CPM, counts per million mapped reads) and low coverage peaks (CPM<1 in more than half the IP samples) were removed. Differential enrichment analysis of read counts was performed using EdgeR.
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text files generated by edgeR that include CPM values in each Sample, logCPMs for all samples, and logFCs, P values and FDR-adjusted P values for the Tex19.1-/- vs Tex19.1+/- comparison.
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| Submission date |
Apr 01, 2019 |
| Last update date |
Apr 01, 2021 |
| Contact name |
Ian Adams |
| Organization name |
MRC Human Genetics Unit
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| Department |
Genome Regulation
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| Street address |
Crewe Road
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| City |
Edinburgh |
| ZIP/Postal code |
EH42XU |
| Country |
United Kingdom |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE129100 |
H3K9me3 ChIP Sequencing of Tex19.1-/- Mouse Embryonic Stem Cells [ChIP-seq] |
| GSE129102 |
H3K9me3 ChIP-Seq and RNA-seq of Tex19.1-/- Mouse Embryonic Stem Cells |
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| Relations |
| BioSample |
SAMN11296830 |
| SRA |
SRX5611044 |
| 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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