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GEO help: Mouse over screen elements for information. |
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Status |
Public on Sep 06, 2022 |
Title |
B cells, mock 4 days, pSTAT3, unstimulated [ChIP-seq] |
Sample type |
SRA |
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Source name |
Spleen
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Organism |
Mus musculus |
Characteristics |
tissue: Spleen strain: C57BL/6J cell type: B cells chip antibody: pSTAT3 (CST D3A7) treatment: Untreated, unstimulated
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Treatment protocol |
Cells were treated with vehicle or with 100 ug/mL L-ascorbic acid 2-phosphate at the beginning of culture
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Growth protocol |
Naïve B cells were co-cultured with irradiated 40LB stromal cells in the presence of recombinant mouse interleukin-4 (1ng/mL; Peprotech) in complete media (RPMI1640, 10% fetal bovine serum, 2mM GlutaMax, 1x non-essential amino acid, 1mM sodium pyruvate, 10mM HEPES, 50 ug/mL gentamicin, and 55 uM 2-mercaptoethanol). Cells were incubated at 37°C with 5% CO2 for 4 days.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were fixed with 1% formaldehyde (ThermoFisher) in media at 1 × 106 cells/ml for 10 mins at room temperature with constant nutation. Fixation was quenched with 125 mM glycine for 5 min on ice. After being washed twice with PBS, cell pellets were snap-frozen with liquid nitrogen and stored at −80 °C until nuclei preparation. To extract chromatin, nuclei were isolated using lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, and 0.25% Triton X-100) and were washed once with wash buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), followed by two washes with shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.1% SDS) without disturbing pellets. Pellets were then resuspended in shearing buffer and the chromatin was sheared using Bioruptor Pico (Diagenode) for 7 cycles (30 sec on, 30 sec off) at 4 °C. After precleared with ProteinA dynabeads (ThermoFisher), 20 μg of chromatin was diluted to a final of 500uL RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.1% SDS, and 0.5% sodium deoxycholate) and incubated with 1 μg rabbit monoclonal anti-phsopho-STAT3 (Tyr705) antibody (clone D3A7, Cell Signaling Technology) overnight at 4°C with constant rotation. To capture the antibody-bound chromatin, the samples were incubated with 30 μl of precleaned Protein A Dynabeads for 2 hours at 4 °C. The beads were washed twice with RIPA buffer and once with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). All washes were incubated for 5 min at 4 °C with constant rotation. After the last wash, samples were eluted twice with 100 μl elution buffer (100 mM NaHCO3,1% SDS, and 0.5 mg/ml RNase A). To decrosslink, proteinase K (0.5 mg/ml; Ambion) and NaCl (200 mM) were added to the eluted samples, followed by the incubation at 65 °C overnight. The samples were purified using DNA Clean & Concentrator-5 (Zymo Research). Sequencing libraries were prepared using NEB Ultra II kits according to the standard protocol (NEB).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Day4_Mock_NoIL21
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Data processing |
Bowtie2 (v2.4.2) Samtools (v1.10) Deeptools (v3.5.1) Assembly: mm10 Supplementary files format and content: Bigwig
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Submission date |
Jun 20, 2022 |
Last update date |
Sep 06, 2022 |
Contact name |
Chan-Wang Jerry Lio |
E-mail(s) |
lio.4@osu.edu
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Organization name |
Ohio State University
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Street address |
460 W 12th Ave
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City |
Columbus |
State/province |
Ohio |
ZIP/Postal code |
43210 |
Country |
USA |
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Platform ID |
GPL24247 |
Series (2) |
GSE183681 |
Epigenetic Remodeling by Vitamin C Potentiates Plasma Cell Differentiation |
GSE206452 |
Vitamin C Potentiates Plasma Cell Differentiation via TET-mediated DNA modification [ChIP-seq] |
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Relations |
BioSample |
SAMN29207428 |
SRA |
SRX15797912 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6254254_VC102_STAT3_MOCK_IL-21_0h-1.bw |
71.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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