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| Status |
Public on Apr 29, 2019 |
| Title |
B6WT_72h_DSG_Tet2 |
| Sample type |
SRA |
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| Source name |
B6WT_72h_DSG_Tet2
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
age: 8-12 weeks
genotype/variation: WT_B6
tissue: Spleen
antibody rrid: AB_2722695
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| Treatment protocol |
Cells were activated with LPS (25ug/mL) and IL-4 (10ng/mL) for 72h
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were centrifugation at 250 x g for 5 mins and cell pellets were resuspended in 37°C PBS with 2mM disuccinimidyl glutarate (ThermoFisher) to crosslink proteins for 30 mins at room temperature. Formaldehyde was added to a final concentration of 1% and the cells were incubated at room temperature for 10 mins. Fixation was quenched with 125 mM glycine and cells were washed twice with ice cold PBS. Cells were pelleted, snap-froze with liquid nitrogen. To isolate nuclei for sonication, cell pellets were thawed on ice and lysed with lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100) for 10 mins at 4°C with rotation, washed once with washing buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and twice with shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS). Nuclei were resuspended in 1mL shearing buffer and sonicated with Covaris E220 using 1 mL milliTUBE (Covaris, Woburn, MA) for 18-20 minutes (Duty Cycle 5%, intensity 140 Watts, cycles per burst 200). After sonication, insoluble debris was removed by centrifugation at 20,000 x g. Buffer for chromatin was adjusted with 1 volume of 2x conversion buffer (10 mM Tris-HCl pH 7.5, 280 mM NaCl, 1 mM EDTA, 1mM EGTA, 0.2% sodium deoxycholate, 0.2% Triton-X100, 1% Halt protease inhibitors with 0.1% SDS). Chromatin was pre-cleared with washed protein A dynabeads (ThermoFisher) for 2 hours, incubated with rabbit polyclonal anti-Tet2 (ab124297; Abcam) and protein A dynabeads overnight (all procedures were at 4°C with rotation). Bead-bound chromatin was washed twice with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS), once with high salt wash buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was eluted from beads with elution buffer (100 mM NaHCO3, 1% SDS, 1 mg/mL RNaseA; Qiagen) twice for 30 mins each at 37°C with constant shaking. NaCl and proteinase K (Ambion) were added to the eluted chromatin at concentrations of 250 mM and 0.5 mg/mL, respectively, and de-crosslinked at 65°C overnight with constant shaking. DNA was purified with Zymo ChIP DNA Clean & Concentrator-Capped Column (Zymo Research, Irvine, CA).
Library was prepared with NEB Ultra II library prep kit (NEB) following manufacture’s instruction.
ChIP-seq, 50bp single end with HiSeq2500
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Single end 50bp reads were mapped to mm10 using bowtie 2 (v2.1.0) with “-very-sensitive”
Mapped SAM files were converted to BAM using Samtools (v1.7) view –h –F 4
Duplicates were removed using Picard (v2.7.1) "MarkDuplicates"
Mapped read counts were obtained using samtools view -b -c
Bam files were converted to bedGraph normalized to read per 10,000,000 reads using bedtools genomecov -bg -scale
bedGraph files were converted to BigWig using bedGraphToBigWig (v4)
Genome_build: mouse genome mm10 GRCm38 (Dec. 2011) from UCSC
Supplementary_files_format_and_content: bigWig file that contains all the reads that map to the genome; reads are normalized to 10M reads
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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) |
| GSE116207 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer [Tet2-ChIP-seq] |
| GSE116208 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer |
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| Relations |
| BioSample |
SAMN09479110 |
| SRA |
SRX4290598 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3213969_JL2018-1-DSG-Tet2_S8_R1_001.trim.rms.bw |
446.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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