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| Status |
Public on Jun 07, 2023 |
| Title |
DN3 thymocytes, Bcl11b WT, rep 2 |
| Sample type |
SRA |
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| Source name |
thymus
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| Organism |
Mus musculus |
| Characteristics |
tissue: thymus
strain: C57BL/6J
age: 3-6 weeks
Sex: male
cell type: DN3 thymocytes
replicate: 2
genotype: Bcl11bf/f; Vav1-Cre-/-
treatment: NA
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq was performed as described previously (Buenrostro et al., Nature Methods, 2013) with minor modifications. For nuclei isolation, 50,000-60,000 FACS-sorted cells were lysed in Thymocyte Hypotonic Buffer (25 mM HEPES, 5 mM MgCl2, 0.05 mM EDTA, 10% Glycerol, 0.1% NP40. Tagmentation reactions were performed for 30 minutes at 37 °C using reagents from Illumina Tagment DNA TDE1 Enzyme and Buffer Kit. Tagmented DNA samples were purified with DNA Clean & Concentrator-5 kit (Zymo Research).
Libraries were quantified by qPCR, amplified accordingly, and purified using DNA Clean & Concentrator kit (Zymo Research).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Adaptor sequences were trimmed using SeqPurge (v2019_11).
Trimmed reads were mapped to mm10 mouse genome assembly using Bowtie2 (v2.2.9) with settings --very-sensitive -X 2000.
PCR duplicates were removed using Picard (v2.21.8) MarkDuplicates REMOVE_DUPLICATES=true VALIDATION_STRINGENCY=LENIENT.
Reads with MAPQ scores below 30 were purged using samtools (v1.9) view with settings -b -q 30 -f 2 -F 1804.
Peaks were called for each sample using MACS2 (v2.2.7.1) with settings --shift -75 --extsize 150 --nomodel --call-summits --nolambda --keep-dup all -p 0.01 --min-length 100. For each sample, a 501 bp fixed-width peak set was generated by extending the MACS2 summits by 250 bp in both directions. Peaks were ranked by significance (MACS2 peak score) and overlapping peaks with lower peak scores were removed iteratively to create non-overlapping sample peak sets. To facilitate comparison of peaks across samples, each peak’s ‘score-per-million’ (SPM) was calculated by dividing the peak score by the sum of all peak scores in the sample, divided by 1 million. All sample peak sets were merged, less significant overlapping peaks removed, and remaining peaks were filtered for those that were observed in at least two samples with an SPM value > 5. We removed peaks mapping to chrY as well as any that spanned genomic regions containing “N” nucleotides. We then removed any peak that overlapped with those called in activated B cell samples to create a “Total T cell” merged peak set. To identify T effector-specific peaks, sample peak sets from only peripheral T effector samples were merged and processed as above for non-overlapping, robust, reproducible peaks purged of activated B cell peaks. To account for less robust but significant T effector peaks which were robustly accessible in early thymocyte development, we added filtered sample peaks from DN1-DN4 stages (reproducible, SPM > 5) that overlapped with reproducible T effector peaks at SPM > 2 to this initial filtered T effector merged set. This merged set was again filtered for non-overlapping, robust (SPM > 5), reproducible peaks purged of activated B cell peaks to generate the final union peak set referred to as “T effector loci”.
To generate peak-by-sample count matrices, ATAC fragment counts within each peak were normalized by the number of inserts intersecting nucleosome-depleted promoter regions (-300 bp to +100 bp relative to transcriptional start-sites).
Assembly: mm10
Supplementary files format and content: tab-delimited text file containing TSS-normalized fragment counts over all T effector loci (n = 65253) for each ATAC-seq sample
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| Submission date |
Jun 06, 2023 |
| Last update date |
Jun 07, 2023 |
| Contact name |
Alexandra Bradu |
| E-mail(s) |
abradu@uchicago.edu
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| Organization name |
University Of Chicago
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| Department |
Pathology
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| Lab |
Koh Lab
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| Street address |
924 East 57th Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE234329 |
PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape [ATAC-seq] |
| GSE234331 |
PU.1 and BCL11B sequentially cooperate with RUNX1 to anchor mSWI/SNF to poise the T cell effector landscape |
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| Relations |
| BioSample |
SAMN35026769 |
| SRA |
SRX20278381 |
| 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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