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| Status |
Public on Apr 25, 2020 |
| Title |
GT_E13_5_InvV_CnR_CTCF |
| Sample type |
SRA |
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| Source name |
genital tubercule
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| Organism |
Mus musculus |
| Characteristics |
genotype: Inv(V)
developmental stage: E13.5
strain: CBA / C57BL/6 mix
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CUT&RUN (Schmid et al., 2004; Skene and Henikoff, 2017) was performed as described in (Meers et al., 2019; Skene et al., 2018). Micro-dissected tissues were isolated in PBS supplemented with 10% fetal calf serum and dissociated to single cell by collagenase treatment. After isolation, 500’000 cells were washed and bound to concanavalin A-coated magnetic beads and permeabilized with wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and Roche Complete protein inhibitor) containing 0.02% digitonin. Bound cells were incubated with primary antibody (anti-HOXA13, AbCam ab106503; anti-CTCF, Active Motif, 6131) for 2h at room temperature. After washes the samples were incubated with Protein A-MNase (pA-MN) for 1 hour at 4oC, then washed twice more with Wash Buffer. Samples were resuspended in low-salt rinse buffer (20 mM HEPES, pH7.5, 0.5 mM spermidine, and 0.125% Digitonin) and chilled to 0°C and the liquid was removed on a magnet stand. Ice-cold calcium incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.05% Digitonin) was added and samples were incubated on an ice-cold block for 30 min. STOP buffer (270 mM NaCl, 20 mM EDTA, 4 mM EGTA ,0.02% Digitonin, 50 µg glycogen, 50 µg RNase A) was added and samples were incubated at 37°C for 30 min, replaced on a magnet stand and the liquid was removed to a fresh tube. DNA was extracted by Phenol-Chloroform extraction and ethanol precipitation. Libraries were prepared as described in (Skene et al., 2018). Library quality was checked on a fragment analyzer, and paired-end sequencing was performed on an Illumina NextSeq 500 instrument (read length 2 × 37 base pairs).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: CUT & RUN
CUT&RUN reads processing was done on the Duboule local Galaxy server (Afgan et al., 2016). Adapters and bad-quality bases were removed with Cutadapt version 1.16 (Martin, 2011). Reads were mapped to the mouse genome (mm10) using Bowtie2 (v2.3.4.1) (Langmead and Salzberg, 2012), (-I 0 -X 1000 --fr --dovetail --very-sensitive). Reads with mapping quality below 30 or not properly paired were removed from the analysis. The output BAM file was converted to BED using bamtobed bedtools v2.18.2 (Quinlan, 2014). The coverage was obtained as the output of MACS2 (v2.1.1.20160309) (Zhang et al., 2008) (--format BED --keep-dup 1 --bdg --nomodel --extsize 200 --shift -100).
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph
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| Submission date |
Oct 07, 2019 |
| Last update date |
Apr 26, 2020 |
| Contact name |
Ana Rita Amandio Lhopitallier |
| E-mail(s) |
rita.lhopitallier@epfl.ch
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| Organization name |
EPFL
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| Department |
SV-ISREC-EPFL
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| Lab |
Laboratory of Developmental Genomics
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| Street address |
SV2842, Station 19
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| City |
Lausanne |
| ZIP/Postal code |
CH-1015 |
| Country |
Switzerland |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE138510 |
A complex regulatory landscape involved in the development of external genitals [CnR] |
| GSE138514 |
A complex regulatory landscape involved in the development of external genitals |
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| Relations |
| BioSample |
SAMN12982912 |
| SRA |
SRX6958329 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4110090_GT_E13_5_InvV_CnR_CTCF.bedgraph.gz |
175.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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