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| Status |
Public on Apr 21, 2020 |
| Title |
control2dpi-3 |
| Sample type |
SRA |
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| Source name |
small intestinal crypts
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| Organism |
Mus musculus |
| Characteristics |
tissue: small intestinal crypts
strain/genotype: Tg(Vil1-cre/ERT2); Ctnnb1-wt/flox
days post cre induction: 2
background strain: C57BL/6
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| Treatment protocol |
To induce Cre-ERT2-mediated recombination, tamoxifen (Sigma, 80 mg/kg) was injected intraperitoneally on two consecutive days. Mice were sacrificed 48 hours after the first tamoxifen injection.
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| Growth protocol |
All animals were kept on a C57BL/6 background. Mice were 8-12 weeks old at the time of treatments and cell isolations. Mice of both sexes were used in all experiments and littermates were used as controls.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Control (n=3) and D164A (n=3) mice were sacrificed 2d pi and processed independently for ATACSeq library preparation, following the protocol by Buenrostro et al, 2015. Briefly, single cell suspension of duodenal crypts was performed as described (Sato and Clevers, 2013). 50’000 cells were lysed in 50ml cold Lysis buffer (10 mM Tris-HCl,pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). After centrifugation at 500 g for 10 min at 4°C, cells were resuspended in 50 mL 1X Illumina transposition mix (25ml Tagment DNA Buffer, 2.5ml Tagment DNA Enzyme, 22.5ml nuclease-free H2O) and incubated for 30 min at 37°C on a shaker. Immediately following the transposition reaction, DNA was purified using the QIAgen MinElute PCR Purification Kit according to the manufacturer’s instructions.
Library was amplified with Nextera Sequencing primers in NEBNext Hot Start High-Fidelity 2X PCR master mix for 5 cycles. The appropriate number of additional PCR cycles was determined by qPCR. Amplified libraries were purified using the QIAgen MinElute PCR Purification Kit. DNA was eluted in 20ml EB buffer and quantified and visualized for quality control with a 2200 TapeStation System (Agilent). Libraries were sequenced on an Illumina HiSeq2500 with paired end 70 bp read configuration.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Adapters trimming with cutadapt, alignment on GRCh38 using Bowtie2 with default mapping parameters (Landmead and Salzberg, 2012), as well as duplicate filtering with Picard and peak calling with MACS2 (p<0.01) were performed on the ENCODE pipeline (Davis et al, 2018) supported by the SUSHI application AtacENCODEApp, for every biological replicate separately.
Differential peaks between sample groups were identified using the exact test function in edgeR. Only peaks with a log fold change > 1 and an p value < 0.01 were considered as differentially accessible for further analysis.
Genome_build: Mus_musculus.GRCm38.95
Supplementary_files_format_and_content: narrowPeak files
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| Submission date |
Apr 20, 2020 |
| Last update date |
Apr 22, 2020 |
| Contact name |
Tomas Valenta |
| E-mail(s) |
tomas.valenta@mls.uzh.ch
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| Organization name |
University of Zurich
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| Department |
Institute of Molecular Life Sciences
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| Street address |
Winterthurerstrasse 190
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| City |
Zurich |
| ZIP/Postal code |
CH-8057 Zurich |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE148940 |
Discrete regulation of β-catenin-mediated transcription governs identity of intestinal epithelial stem cells [ATAC-Seq] |
| GSE148943 |
Discrete regulation of β-catenin-mediated transcription governs identity of intestinal epithelial stem cells |
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| Relations |
| BioSample |
SAMN14645685 |
| SRA |
SRX8142697 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4486355_20200129.control_3.narrowPeak.gz |
2.6 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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