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| Status |
Public on Jun 30, 2020 |
| Title |
Brain_Endothelium_SteadyState_CHIP_2 |
| Sample type |
SRA |
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| Source name |
Murine structural cells
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| Organism |
Mus musculus |
| Characteristics |
organ: Brain
cell type: GP38negCD31pos
condition: steady_state
age: 8 to 12 weeks
strain: C57BL/6J
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| Growth protocol |
C57BL/6J mice , bred and maintained under specific pathogen free conditions at the Insti-tute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences in Vienna, Austria. 8 to 12 week old mice (males only ) were used for the steady state characterization
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Organs were digested with Accumax and structural cells isolated using FACS
ChIPmentation was carried out as previously described (Gustafsson et al., 2019; Schmidl et al., 2015), with minor adaptions. Briefly, 50,000 cells (or a lower number of cells for particularly rare cell populations) were washed once with PBS. Cells were fixed and sonicated (Covaris S220) as described (Schmidl et al., 2015). Following sonication, the lysate was brought to RIPA buffer conditions (final concentration: 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1x protease inhibitors (Sigma) and 1 mM PMSF) to a volume of 200 µl per immunoprecipitation. For each immunoprecipitation, 10 µl magnetic Protein A (Life Technologies) were washed twice and resus-pended in PBS supplemented with 0.1% BSA. 1 µg of antibody recognizing H3K4me2 (MerckMillipore, clone AW30) was added and bound to the beads by rotating over night at 4 °C. Beads were added to the sonicated lysate and incubated for 2 h at 4 °C on a rotator followed by washing the beads and tagmenting the bead-bound chromatin as described (Gustafsson et al., 2019). After tagmentation, the beads were washed once with RIPA and once with cold Tris-Cl pH 8. Bead bound tagmented chromatin was than resuspended in 10.5 µl 20 mM EDTA and incubated for 30 min at 50 °C. Then, 10.5 µl 20 mM MgCl2 as well as 25 µl of pre-activated 2x KAPA HiFi HotStart Ready Mix (incubation at 98 °C for 45 s) are added and incubated for 5 min at 72 °C followed by incubation for 10 min at 95 °C. Beads are magnetized and 2 µl of each library were amplified in a 10 µl qPCR reaction containing 0.8 mM primers, SYBR Green and 5 µl Kapa HiFi HotStart ReadyMix to estimate the optimum number of enrichment cycles with the following program: 72 °C for 5 min, 98 °C for 30 s, 24 cycles of 98 °C for 10 s, 63 °C for 30 s and 72 °C for 30 s, and a final elongation at 72 °C for 1 min. Kapa HiFi HotStart ReadyMix was incubated at 98 °C for 45 s before preparation of all PCR reactions (qPCR and final enrichment PCR), to activate the hot-start enzyme for successful nick translation at 72 °C in the first PCR step. Final enrichment of the libraries was performed in a 50 µl reaction using 0.75 mM primers (custom Nextera primers as described for ATAC-seq) and 25 ml Kapa HiFi HotStart ReadyMix. Libraries were amplified for the rounded-down Cq value determined in the qPCR reaction. Enriched libraries were purified using SPRI AMPure XP beads. For preparation of compatible input controls, 3 μl of 50 mM MgCl2 was added to 15 μl sonicated lysate (pool of 5 μl of endothelium, epithelium and fibroblasts lysates form the same organ) to neutralize the EDTA in the SDS lysis buffer. 20 μl of tagmentation buffer and 1 μl transposase (Nextera, Illumina) was added, and samples were incubated at 37 °C for 10 min. Input control chromatin was purified with MinElute PCR purification kit (Qiagen) and 22.5 μl of the purified transposition reaction were combined with 25 μl of PCR master mix and 0.75 mM primers (custom Nextera). Libraries were subsequently amplified as described above. The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 50-bp single-end configuration
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
H3K4me2 bound DNA fragments (ChIPmentation)
ChIPmentation sample of Brain GP38negCD31pos cells
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| Data processing |
Adapter trimming with trimmomatic (version 0.32) with settings LEADING:3 TRAILING:3 SLIDINGWINDOW:4:10 MINLEN:36
Genome alignment using bowtie (2.2.4) with settings --very-sensitive
Filtered reads to primary alignments with MAPQ > 30 using sambamba (0.7.0)
Called peaks using MACS2 (2.7.6)
Counted reads in consensus peak set over all samples using GenomicAlignments in R (3.2.3)
Normalized to counts per million (CPM) and transformed to log2(CPM) using Limma Voom in R
Regressed out immune cell signatures using Limma removeBatchEffect in R
Genome_build: mm10
Supplementary_files_format_and_content: Matrix of read counts (CSV), read counts of each peak in the consensus peak set for each sample
Supplementary_files_format_and_content: Matrix of log2(CPM) values (CSV), log2(CPM) after regressing out immune signatures of each peak in the consensus peak set for each sample
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| Submission date |
Mar 06, 2020 |
| Last update date |
Jun 30, 2020 |
| Contact name |
Christoph Bock |
| E-mail(s) |
cbock@cemm.oeaw.ac.at
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| Organization name |
CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences
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| Street address |
Lazarettgasse 14
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE134658 |
Structural cells are key regulators of organ-specific immune response |
| GSE134663 |
Structural cells are key regulators of organ-specific immune response |
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| Relations |
| BioSample |
SAMN14322498 |
| SRA |
SRX7866270 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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