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Status |
Public on Apr 29, 2019 |
Title |
Sample 16_WT.24.IP.E2 |
Sample type |
SRA |
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Source name |
Tet2/3-WT_24h_CMSIP
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Organism |
Mus musculus |
Characteristics |
strain background: C57BL/6J age: 8-12 weeks genotype/variation: Tet2/3-WT (Tet2-flox, Tet3-flox, Rosa26-LSL-YFP) tissue: Spleen molecule subtype: Bisulfite-treated genomic DNA
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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 |
Genomic DNA isolated from naïve and activated B cells was spiked with unmethylated lambda phage cI857 Sam7 DNA (Promega, Madison, WI) and a PCR amplicon from puromycin resistant gene at a ratio of 200:1 and 100,000:1, respectively. DNA (5-10 ug in 130 uL TE buffer) was sheared with a Covaris E220 (Covaris) using microTUBE for 4 mins. DNA was cleaned-up with AmureXP beads, processed with NEBNext End Repair and A-tail Modules (NEB, Ipswich, MA), and ligated to methylated Illumina adaptors (NEB). DNA was then bisulfite-treated, denatured, and immunoprecipitated with anti-CMS serum (in house) and mixture of protein A and G dynabeads (ThermoFisher). Libraries for immunoprecipitated DNA were generated by PCR with barcoded primers (NEBNext Multiplex Oligos for Illumina, NEB) for 15 cycles using KAPA HiFi HotStart Uracil+ ReadyMix (Roche), followed by a cleanup with AmpureXP beads (Beckman Coulter). Bisulfite-seq, 50bp paired-end with HiSeq 2500
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Library strategy |
Bisulfite-Seq |
Library source |
genomic |
Library selection |
RANDOM |
Instrument model |
Illumina HiSeq 2500 |
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Description |
5hmC-enriched by CMSIP 2-7-17-Jlio-CMSIP-09_S17
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Data processing |
Bcl2fastq v2.20.0.442 was used to obtain fastq files from Illumina Paired-end reads (50bp) were mapped to the mouse genome mm10 GRCm38 (Dec. 2011) from UCSC, using BSMAP (V.2.74) (-v 4 -R -n 1 -w 2 -r 0 -q 20 -R -p 8). Reads that mapped to the spike-in control (chrPhagueLambda and Puro) were filtered out from the sam file using awk. Tag directories were created with the remaining reads using makeTagDirectory from HOMER (-genome mm10 -tbp 1 –checkGC). Peaks were called with findPeaks from HOMER (-style histone -o auto -i). Quantile normalization was applied to all raw counts files and differentially enriched 5hmC regions were identified with edgeR (Robinson et al., 2010); a p adjusted value of <= 0.05 Genome_build: mouse genome mm10 GRCm38 (Dec. 2011) from UCSC Supplementary_files_format_and_content: Quantile normalized counts, in a csv format. Column1: chromosome; column2:start; column3:end; coulmn4-27:Samples1-24
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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) |
GSE116201 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer [CMS-IP] |
GSE116208 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer |
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Relations |
BioSample |
SAMN09479081 |
SRA |
SRX4290533 |
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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