|
| Status |
Public on May 07, 2020 |
| Title |
WT_H3K27ac_ChIPSeq_spikein_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
limb buds mouse embryo
|
| Organism |
Mus musculus |
| Characteristics |
tissue: limb bud
developmental state: E12.5
genotype: wild-type
chip antibody: H3K27ac (Diagenode, C15410174)
|
| Treatment protocol |
Limb buds (stage E12.5) were micro-dissected from mouse embryos treatment protocol.
|
| Growth protocol |
E12.5 littermate embryos (wt and homozygous) were obtained from staged matings of heterozygous Spdh mice.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Dissected tissue from embryos was pooled and turned into a single-cell suspension by digestion with Trypsin-EDTA 0.05% (Gibco) for 10 min at 37°C shaking at 900 RPM. Drosophila melanogaster S2 cells were spiked in in a 1:3 ratio. The cells were mixed with 10%FCS/PBS and homogenized using a 40 µm cell strainer (Falcon). After centrifugation, cells were fixed in 1% PFA/10%FCS/PBS for 10’ at room temperature. Cells were then lysed 3C-Lysis buffer (50mM TRIS pH 7.5; 150mM NaCl; 5mM EDTA; 0.5% NP-40; 1.15% Triton X-100; Protease Inhibitors (Roche)) and nuclei pelleted by centrifugation. For sonication, nuclei were resuspended in buffer 3 (Lee et al. 2006). Chromatin was sheared using a Bioruptor until reaching a fragment size of 200-500bp. Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with the respective antibody. 10-15μg of chromatin was used for each replicate ChIP. Antibodies: H3K27ac (Diagenode, C15410174) as in Ibrahim et al. 2013.
Sequencing libraries were prepared using Nextera adaptors.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
ChIPseq
|
| Data processing |
Basecalls performed using CASAVA version 1.4
Paired-end ChIP-Seq reads were quality filtered and adapter trimmed by using cutadapt version 1.18 with minquality 20, minlength 25 and illumina adapter sequence AGATCGGAAGAGC
Reads were aligend to the mouse genome mm9 or fly (D. melanogaster) genome dm6 with bwa version 0.7.17-r1188 using the ‘mem’ command and default options. A sorted bam file and corresponding index was created with samtools version 1.9
Duplicate reads have been identified and filtered by using MarkDuplicates from the gatk suite version 4.1.4.0.
Deduplicated reads were kept if their MAPQ value was at least 15 by using samtools with option -q 15
Peaks for the mapped mouse reads/fly reads were called using the callpeak program from macs2 version 2.1.2 and using INPUT ChIP as control and the option ‘narrow’.
Technical replicates (samples 7,8 and 9,10) were merged using the merge program from bamtools version 2.5.1.
Genome wide rpm per bp normalized coverage profiles were created by using bamToGFF_turbo.py from https://github.com/BradnerLab/pipeline.git. The genome was divided into 3mb regions and the tool was run with options -s both -e 200 -m 60000 resulting in 50bp bins.
Wig files from the rpm per bp matrices were created with samtools and then multiplied with dm6 spikein normalization factors
Genome_ build: mm9, dm6
Supplementary_files_format_and_content: bigwig files with spikein normalized rpm per bp values were created using samtools
|
| |
|
| Submission date |
Dec 21, 2019 |
| Last update date |
May 07, 2020 |
| Contact name |
Denes Hnisz |
| E-mail(s) |
hnisz@molgen.mpg.de
|
| Organization name |
Max Planck Institute for Molecular Genetics
|
| Department |
Genome Regulation
|
| Lab |
Hnisz Lab
|
| Street address |
Ihnestraße 63-73
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE128818 |
Aberrant phase separation of transcription factors in human developmental disorders |
|
| Relations |
| BioSample |
SAMN13663900 |
| SRA |
SRX7427594 |
