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| Status |
Public on Apr 29, 2019 |
| Title |
Sample 24_WT.72.IP.E3 |
| Sample type |
SRA |
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| Source name |
Tet2/3-WT_72h_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
WT-72-CMS_S32
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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 |
SAMN09479106 |
| SRA |
SRX4290541 |
| 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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