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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 29, 2019 |
| Title |
DKO_00h_rep2 |
| Sample type |
SRA |
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| Source name |
Tet2/3-DKO_00h_ATAC
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
age: 8-12 weeks
genotype/variation: Tet2/3-DKO (CreERT2, Tet2-flox, Tet3-flox, Rosa26-LSL-YFP)
tissue: Spleen
molecule subtype: Tn5 (ATAC)
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| Treatment protocol |
Cells were either unstimulated or activated with LPS (25ug/mL) and IL-4 (10ng/mL)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
50,000 cells were collected by centrifugation and washed once with 50uL ice-cold PBS and centrifuged at 600 xg for 5 mins at 4°C. Cell pellets were resuspended in 50 uL of cold lysis buffer (10mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and spin down immediately at 500 xg for 10 mins at 4°C. Supernatant was discarded and nuclei were resuspended in 50uL transposition reaction mix (25uL 1x TD buffer fom Illuminia, 2.5 uL Tn5 transposase, 22.5 uL H2O), incubated at 37°C for 30 mins, and DNA was purified with a Qiagen MinElute kit (Qiagen).
Library was amplified with KAPA HiFi HotStart Real-time PCR Master Mix (Roche) using indexed primers.
ATAC-seq, 50bp paired-end with HiSeq2500
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
11-18-16-JerryATACseq4_S4
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| Data processing |
ATAC-seq Paired-End reads were mapped to the mouse genome mm10 GRCm38 (Dec. 2011) from UCSC using bowtie 1.0.0 ("-p 8 -m 1 --best --strata -X 2000 -S --fr --chunkmbs 1024.").
Reads that failed this alignment step were filtered for Illumina adapters and low quality using trim_galore ("--paired --nextera --length 37 --stringency 3 --three_prime_clip_R1 1 --three_prime_clip_R2 1") and re-mapped using the same parameters.
Both mapping resulst were merged and processed together to remove duplicates using picard-tools-1.94 MarkDuplicates; Mitochondrial and Chromosome Y reads were excluded as well.
Subnucleosomal fragments were obtained with samtools and awk ("'{if(sqrt(\$9*\$9)<100){print \$0}}’") to identify DNA fragments that were less equal than 100 nt in length.
These fragments were used to call peaks using HOMER (v4.9.1) findPeaks function per replicate ("-size 500 -region -center -P .1 -LP .1 -poisson .1 -style dnase") and all the peak sets were merged to generate a global set.
Peaks overlapping with ENCODE blacklisted regions (Consortium, 2012; Kundaje, 2013) were removed.
From each sample, Tn5 insertion sites were obtained by isolation of the initial 9bp of mapped reads (Buenrostro et al., 2013) which were used to compute the number of transposase insertions per peak using MEDIPS (Chavez et al., 2010).
Before differential coverage calculation, all of the raw reads were quantile normalized altogether.
For differential coverage, the quantile normalized data was given as input to edgeR, avoiding the TMM normalization to re-normalize, and incuding only those regions that added up to more than 32 normalized reads across the samples per comparison.
Differentially accessible regions were defined by an adjusted p value (FDR) lower than 0.05 and a log 2 fold enriichment higher equal than 1.
Genome_build: mouse genome mm10 GRCm38 (Dec. 2011) from UCSC
Supplementary_files_format_and_content: Quantile Normalized Tn5 Insertion sites per sample on all the set of peaks (space-delimited)
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| Submission date |
Jun 25, 2018 |
| Last update date |
Apr 29, 2019 |
| Contact name |
Anjana Rao |
| E-mail(s) |
arao@lji.org
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| Organization name |
La Jolla Institute for Allergy and Immunology
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| Department |
Signaling and Gene Expression
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| Lab |
Anjana Rao
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| Street address |
9420 Athena Cir
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE116203 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer [ATAC-seq] |
| GSE116208 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer |
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| Relations |
| BioSample |
SAMN09479143 |
| SRA |
SRX4290559 |
| 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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