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| Status |
Public on Aug 26, 2022 |
| Title |
Host WT conventional T cells, rep2 |
| Sample type |
SRA |
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| Source name |
Lymphocytes
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| Organism |
Mus musculus |
| Characteristics |
strain: Balb/c
cell type: T cell
sorting markers: CD45.1+CD45.2-CD3+CD4+CD25-
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| Growth protocol |
Mice were housed at UKR central animal facility
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin accessibility mapping on purified cell populations was performed using the ATAC-seq method as previously described (Buenrostro et al., 2013; Corces et al., 2017), with minor adaptations. Briefly, in each experiment 2,000 - 15,000 sorted cells were pelleted by centrifuging for 10 min at 4 °C at 500 x g. After centrifugation, the pellet was carefully lysed in 50 µl resuspension buffer supplemented with NP-40 (Sigma), Tween-20 and Digitonin (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 % NP-40, 0.1 % Tween-20, 0.01 % Digitonin) and incubated for 3 minutes on ice. Then, 1 ml of ice-cold resuspension buffer supplemented with Tween-20 was added, and the sample was centrifuged at 4 °C at 500 x g for 10 minutes. The supernatant was discarded, and the cell pellet was carefully resuspended in the transposition reaction (25 µl 2 x TD buffer (Illumina), 2.5 µl TDE1 (Illumina), 16.5 µl PBS, 5 µl nuclease-free water, 0.5 µl 1% Digitonin (Promega), 0.5 µl 10% Tween-20 (Sigma)) for 30 min at 37 °C on a shaker at 1000 rpm.
Following DNA purification with the Clean and Concentrator-5 kit (Zymo) eluting in 23 µl, 2 µl of the eluted DNA was used in a quantitative 10 µL PCR reaction (1.25 µM forward and reverse custom Nextera primers (Corces et al., 2017), 1x SYBR green final concentration) to estimate the optimum number of amplification cycles with the following program 72°C 5 min; 98°C 30 s; 25 cycles: 98°C 10 s, 63°C 30 s, 72°C 1 min; the final amplification of the library was carried out using the same PCR program and the number of cycles according to the Cq value of the qPCR. Library amplification using custom Nextera primers was followed by SPRI size selection with AmpureXP beads to exclude fragments larger than 1,200 bp. DNA concentration was measured with a Qubit fluorometer (Life Technologies). The libraries were sequenced using the Illumina HiSeq3000/4000 or NextSeq platforms.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
HTconv_R3_CD45-1Pos_CD25Neg_Aire-HCO
HTconv_ATACseq_omniATAC1_50000_illuminaTn5_R3_CD45-1plusCD45-2minusCD3plusCD4plusCD25minus_AireminusHCO_BW13s6_S4
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| Data processing |
Sequenced reads were trimmed for adaptor sequences using skewer v0.2.2
Reads were mapped to mm10 whole genome using bowtie2 v2.3.3 with the –very-sensitive parameter and a maximum insert size of 2000 bp
Duplicate and unpaired reads were removed using the sambamba (Tarasov et al., 2015) ‘markdup’ command, and reads with mapping quality >30 and alignment to the nuclear genome were kept.
Peak calling for each sample was performed using HOMER v4.10 (Heinz et al., 2010) with the following approach: Two different peak sets were called, once we used the options ‘-style factor -fragmentLength 150 -size 250 -minDist 250 –L 4 –fdr 0.0001’ parameters, to identify highly robust small regions of open chromatin, and in a second approach we used the parameters ‘-region -fragmentLength 150 -size 150 -minDist 250 -L 4 -fdr 0.0001’ to identify larger regions of open chromatin. The two peak sets were then merged using the mergePeaks function of HOMER to create the peak set that was used for downstream analysis.
For visualization purposes only, coverage files from filtered bam files were produced using bedtools (Quinlan and Hall, 2010) genomeCoverageBed, each position was normalized by dividing to the total library size and multiplying by 106, followed by conversion to a bigwig using the bedGraphToBigWig command from the UCSC genome browser tools.
Genome_build: mm10
Supplementary_files_format_and_content: Processed data files contain bed files (peaks) and bigWig files (for genome browser visuaizations)
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| Submission date |
Aug 29, 2019 |
| Last update date |
Aug 26, 2022 |
| Contact name |
Philipp Beckhove |
| E-mail(s) |
philipp.beckhove@rcii.de
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| Organization name |
Regensburg Center for Interventional Immunology
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| Street address |
Franz-Josef-Strauss-Allee 11
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| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE136648 |
Evaluation of Genome-wide chromatin accessibility of BM T cell subsets [ATAC_TC] |
| GSE136650 |
Induction of autoreactive regulatory T cells through promiscuous gene expression by bone marrow-resident Aire+ antigen-presenting cells |
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| Relations |
| BioSample |
SAMN12659574 |
| SRA |
SRX6778395 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4054540_HTconv_R3_CD45-1Pos_CD25Neg_Aire-HCO.bigWig |
399.9 Mb |
(ftp)(http) |
BIGWIG |
| GSM4054540_HTconv_R3_CD45-1Pos_CD25Neg_Aire-HCO.intersect.peaks.bed.gz |
549.2 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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