|
| Status |
Public on Dec 06, 2016 |
| Title |
RAD21-HL-E115-Wt-Mm-Rep2-L8226-1 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic Limb Bud
|
| Organism |
Mus musculus |
| Characteristics |
tissue: hindlimb
developmental stages: E11.5
strain: CD1
chip antibody: RAD21 (Abcam: ab992)
|
| Treatment protocol |
For chromatin modifications, chromatin was prepared from the different tissues with a 1% FA crosslinking for 15 minutes and resuspended in buffer 3 for sonication (Lee et al.,). For CTCF and RAD21 ChIP-seq we prepared chromatin as follow: tissues were disrupted in 0.1% collagenase at 37°C and homogenized using a needle. Cell were then centrifuged and resuspended in (10%FCS, 0.2%Cs, 1% L-Glu, 0.5% Pen-Strep in DMEM:HAM’s F-12 1:1)) and fixed in 1% FA for 10’ on ice. Cell were then lysed in buffer 1 and 2 and resuspended in buffer 3 for sonication (Lee et al. 2006).
|
| Growth protocol |
Limb bud and midbrain were micro-dissected from mouse embryos
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
We sheared chromatin using Bioruptor until reaching a fragment size of 200-500bp. 10-15μg of chromatin was then used for each replicate chromatin modification ChIP and 30μg for CTCF and RAD21 ChIP. ChIP for H3K4me1 (Abcam: 8898), H3K4me3 (Milipore: 07-473), H3K27Ac (Diagenode: C1540174), H3K27me3 (Milipore: 07-449), CTCF (Active motif: 613111) and RAD21 (Abcam: ab992) was then performed as in Lee et al., 2006. Libraries were prepared using the Nextera adaptors and sequenced.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
The merge-qfrags.bw file for this sample is available on GSM2251510
|
| Data processing |
Single-end reads from ChIP-seq experiments were mapped with Bowtie-2.2.6 to reference genome mm9. Mapped reads were filtered for mapping quality ≥10, and duplicates were removed.
Reads were extended to a length of 300bp and a scaled (one million / total of unique reads) coverage was computed using genomeCoverageBed from bedtools. We then produced the bigwig files using bedGraphToBigWig tool from UCSC.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
Jul 25, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Guillaume Andrey |
| E-mail(s) |
guillaume.andrey@unige.ch
|
| Phone |
+41223795703
|
| Organization name |
University of Geneva
|
| Department |
Department of Genetic Medicine and Development
|
| Street address |
Rue Michel-Servet 1
|
| City |
Geneva |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE84793 |
Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding [ChIP-seq] |
| GSE84795 |
Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding |
|
| Relations |
| BioSample |
SAMN05438954 |
| SRA |
SRX1975356 |
