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| Status |
Public on Aug 24, 2022 |
| Title |
input_br1 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
condition: normoxia
chip antibody: none
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin extracts were prepared as described in (Brookes et al., 2012). Briefly, cells were fixed with 1% formaldehyde (Thermo, 28906) in PBS at RT for 10 minutes. Then, 0.125M glycine (Sigma, 50046) was added to quench formaldehyde at RT for 5 minutes. Cells were washed twice with ice-cold PBS. To lyse, fixed cells were treated with swelling buffer (25mM HEPES pH 7.9, 1.5mM MgCl2, 10mM KCl (Invitrogen, AM9640G), 0.1% Igepal 630, 1x protease inhibitor cocktail, 1mM PMSF, 2mM NaVO3, 5mM NaF) at 4°C for 10 minutes. Cells were scraped on ice and passed thought 18G needles before centrifugation at 3000g, 4°C for 5 minutes. Cell pellet, corresponding to nuclei was carefully resuspended in sonication buffer (50mM HEPES pH 7.9, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium-deoxycholade (Thermo, 89904), 0.1% SDS (Invitrogen, AM9822), 1mM PMSF, 2mM NaVO3, 5mM NaF) and incubated on ice for 10 minutes before sonication. Chromatin was sheared to an average size of 200-300bp with E220 Evolution Covaris sonicator for 6 cycles, 1 minute each. Shearing efficiency was checked by agarose gel
Library preparation was performed using KAPA Hyper Prep Kit (Kapa Biosystems, KR0961) (input amount 10ng DNA, adapter concentration 1.5 µM and size selection of 200-700-bp after PCR with cycle number = 15) and following manufacturer's instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw reads of treatment and input samples were subjected to adapter and quality trimming with cutadapt (Martin, 2011) (version 2.4; parameters: --nextseq-trim 20 --overlap 5 --minimum-length 25 --adapter AGATCGGAAGAGC -A AGATCGGAAGAGC).
Reads were aligned to the mouse genome (mm10) using BWA with the ‘mem’ command (version 0.7.17, default parameters).
A sorted BAM file was obtained and indexed using samtools with the ‘sort’ and ‘index’ commands (version 1.10). Duplicate reads were identified and removed using GATK (version 4.1.4.1) with the ‘MarkDuplicates’ command and default parameters.
Peaks were called with reads aligning to the mouse genome only using MACS2 ‘callpeak’ (version 2.1.2; parameters --bdg --SPMR) using the input samples as control samples. For identification of consistent peaks, only those that were identified in both replicates were used for downstream analyses. Peaks were annotated using ChIPseeker package (version 1.20.0) using default parameters (TSS region ±3-Kb).
For downstream analyses, after validation of reproducibility, replicates were pooled using samtools ‘merge’. Genome-wide coverage tracks (signal files) for merged replicates normalized by library size were computed using samtools bamCoverage (version 3.4.3) (parameters: --normalizeUsing CPM --extendReads).
Genome_build: mm10
Supplementary_files_format_and_content: bed files containing narrow peaks for each biological replicate
Supplementary_files_format_and_content: bigwig files of genome-wide coverage tracks for merged replicates and normalized by library size (CPM).
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| Submission date |
Jan 27, 2022 |
| Last update date |
Aug 24, 2022 |
| Contact name |
Natalia Lopez-Anguita |
| E-mail(s) |
anguita@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Bulut-Karslioglu Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE178628 |
Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids |
| GSE195545 |
Hypoxia induces an early primitive streak signature, enhancing spontaneous elongation and lineage representation in gastruloids |
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| Relations |
| BioSample |
SAMN25332633 |
| SRA |
SRX13945549 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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