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| Status |
Public on Apr 29, 2019 |
| Title |
WT.0.Input.E2 |
| Sample type |
SRA |
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| Source name |
Tet2/3-WT_0h_input
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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
time point: 0h
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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 |
Cells were fixed with 1% formaldehyde (ThermoFisher) at room temperature for 10 mins at 1E6 cell/mL in media, quenched with 125 mM glycine, 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-H3K27Ac (ab4729, 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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| Description |
Input-Stimulated-WT_S21
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| Data processing |
Bcl2fastq v2.20.0.442 was used to obtain fastq files from Illumina
Single end reads (50bp) were mapped to the mouse genome mm10 GRCm38 (Dec. 2011) from UCSC with Bowtie (V.1.1.2).
Reads were sorted and PCR duplicates were removed using SortSam and MarkDuplicates, respectively from Picard Tools (V.2.7.1).
Tag directories were created with makeTagDirectory (-genome mm10 –checkGC) from HOMER, and peaks were called with findPeaks (-region).
Peaks from all samples were merged with mergePeaks from HOMER into a master table.
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: Tab separated file with the quantile normalized counts
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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) |
| GSE116206 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer [H3K27ac_ChIP-seq] |
| GSE116208 |
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer |
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| Relations |
| BioSample |
SAMN09479115 |
| SRA |
SRX4290593 |
| 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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