|
| Status |
Public on May 05, 2022 |
| Title |
ChIP_Rad21_DistalLimb_E11-5_FALL_r2 |
| Sample type |
SRA |
| |
|
| Source name |
E11.5 distal limb buds
|
| Organism |
Mus musculus |
| Characteristics |
genotype: F-ALL
age: E11.5
tissue: distal limb buds
|
| Treatment protocol |
Distal limbs from E11.5 embryos were fixed in 1% fixation solution (1%PFA in 10%FCS/1XPBS) for 10 minutes at room temperature while tumbling. The reaction was then quenced on ice by adding Glycin (final concentration 125mM). Samples were then centrifuged at 4°C for 8 minutes at 400 g, then twice washed with cold 1X PBS and finally snap frozen and stored at -80 °C.
|
| Growth protocol |
E11.5 mouse limbs were microdissected in 1X PBS and the distal parts of the limb buds were collected and further processed.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The tissues were thawed on ice and resuspended in Chromatin Prep Buffer (High Sensitivity Chromatin Preparation kit (Catalog N. 53046) from Active Motif) with 1x Protease Inhibitor (Roche); nuclei were released using a dounce homogenizer with a tight pestel (Active Motif Ref. 40415) and centrifuged at 4°C at 1250g for 5 minutes. Nuclei were then resuspended in cold Sonication Buffer (0.25% SDS, 10mM Tris-HCl pH 8.0, 2mM EDTA, 1X Protease inhibitor) and pipetted to facilitate nuclei disruption. The chromatin was sonicated using Diagenode Bioruptor (12 cycles 20´´on 30´´off) to achieve a fragment size ranging from 200-500 bp. Sonicated chromatin was incubated overnight with pre-washed A Dynabeads (Thermofisher Cat. N. 10001D) (previously blocked with 0.1% BSA) and 4μg of RAD21 (Ab992) or 1μg of CTCF (Diagenode, #C15410210). After the washes, samples were tagmented in Tagmentation Solution (0.25% Tagmentation Buffer, 2mM Tn5 from Illumina 20034197) for 2 minutes at 37°C. Later the chromatin was eluted in ChIP Elution Buffer (10mM Tris-HCl pH8.0, 5mM EDTA pH8.0, 300mM NaCl, 0.4% SDS). Samples were de-crosslinked purified using AmPure Beads (NEB).
Libraries were prepared using KAPA HiFi HotStart ReadyMix (Roche) and indexed primers from Schmidl et al. 2015. Libraries were purified using AmPure Beads (NEB) and sequenced with 30 millions of single end reads of 75 nt, on NextSeq500 platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIP-seq against Rad21 in developing distal limbs of FALL mice at E11.5
|
| Data processing |
Base calling with standard Illumina Software
Single-end reads were mapped to the same custom reference genomes as specified in the Capture-C section or to mm9 using Bowtie (Langmead et al. 2009, flags -m1 -S --chunkmbs 500). Reads aligning to more than one location were filtered out by bowtie due to the -m1 flag.
Resulting alignments in SAM format were then converted to bam, sorted and deduplicated using samtools (https://github.com/samtools/samtools).
Deduplicated bam files were converted to normalized coverage tracks in bigwig format. They were generated using deepTools BamCoverage (https://github.com/deeptools) after centering the read locations and extending the signal 300bp (bamCoverage -e 300 --centerReads --normalizeUsing RPGC --effectiveGenomeSize 2620345972)
Genome_build: Custom genome based on the mm9 assembly / mm9
Supplementary_files_format_and_content: bigWig
|
| |
|
| Submission date |
Feb 28, 2022 |
| Last update date |
May 05, 2022 |
| Contact name |
Rafael Domínguez Acemel |
| E-mail(s) |
rdacemel@gmail.com
|
| Organization name |
Max Delbrück Center for Molecular Medicine (MDC)
|
| Department |
Berlin Institute of Medical Systems Biology (BIMSB)
|
| Lab |
AG Lupiañez
|
| Street address |
Hannoversche Str. 28
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE169561 |
In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation |
| GSE197629 |
In vivo dissection of a clustered-CTCF domain boundary reveals developmental principles of regulatory insulation [ChIPmentation] |
|
| Relations |
| BioSample |
SAMN26317617 |
| SRA |
SRX14321722 |
