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Status |
Public on Jan 01, 2020 |
Title |
Treg_Klrg1-GFP-_2 [ATAC-seq] |
Sample type |
SRA |
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Source name |
Treg_Klrg1-GFP-_2
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Organism |
Mus musculus |
Characteristics |
cell type: Treg_Klrg1-GFP-
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Growth protocol |
Mice were housed at DKFZ or 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 HiSeq 4000 or NextSeq platforms.
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Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 4000 |
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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 |
May 07, 2019 |
Last update date |
Jan 02, 2020 |
Contact name |
Michael Delacher |
E-mail(s) |
delacher@uni-mainz.de
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Phone |
+49 6131 17 6574
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Organization name |
University Medical Center of the Johannes Gutenberg-University Mainz
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Department |
Institute for Immunology
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Lab |
Immunology
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Street address |
Langenbeckstrasse 1
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City |
Mainz |
State/province |
Rhineland-Palatinate |
ZIP/Postal code |
55131 |
Country |
Germany |
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Platform ID |
GPL21103 |
Series (2) |
GSE130825 |
Identification of tissue regulatory T cell precursors in lymphoid organs [ATAC-Seq] |
GSE130884 |
Identification of tissue regulatory T cell precursors in lymphoid organs |
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Relations |
BioSample |
SAMN11587077 |
SRA |
SRX5802482 |
Supplementary file |
Size |
Download |
File type/resource |
GSM3754904_Treg_Klrg1-GFP-_2.bigWig |
351.9 Mb |
(ftp)(http) |
BIGWIG |
GSM3754904_Treg_Klrg1-GFP-_2.intersect.peaks.bed.gz |
237.6 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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