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Sample GSM6254259 Query DataSets for GSM6254259
Status Public on Sep 06, 2022
Title B cells, VC 4 days, pSTAT3, IL-21 6h, bio rep 2 [ChIP-seq]
Sample type SRA
 
Source name Spleen
Organism Mus musculus
Characteristics tissue: Spleen
strain: C57BL/6J
cell type: B cells
chip antibody: pSTAT3 (CST D3A7)
treatment: VC, IL-21 6h
Treatment protocol Cells were treated with vehicle or with 100 ug/mL L-ascorbic acid 2-phosphate at the beginning of culture
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.
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).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
 
Description Day4_VC_IL21_6h_bio_rep2
Data processing Bowtie2 (v2.4.2)
Samtools (v1.10)
Deeptools (v3.5.1)
Assembly: mm10
Supplementary files format and content: Bigwig
 
Submission date Jun 20, 2022
Last update date Sep 06, 2022
Contact name Chan-Wang Jerry Lio
E-mail(s) lio.4@osu.edu
Organization name Ohio State University
Street address 460 W 12th Ave
City Columbus
State/province Ohio
ZIP/Postal code 43210
Country USA
 
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]
Relations
BioSample SAMN29207423
SRA SRX15797917

Supplementary file Size Download File type/resource
GSM6254259_VC102_STAT3_VC_IL-21_6h-2.bw 58.4 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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