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Sample GSM6935085 Query DataSets for GSM6935085
Status Public on Apr 28, 2023
Title M1_D8_K27Ac_S14
Sample type SRA
 
Source name In vitro differentiated Th9 (d8)
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: In vitro differentiated Th9 (d8)
Treatment protocol Murine naïve CD4+CD62L+ cells were isolated from spleen and lymph nodes using negative selection (Miltenyi) followed by flow cytometric sorting to >95% purity. Cells were cultured at a density of 0.5 x10^6 per mL and activated with plate bound aCD3Ɛ (10 µg/mL) and aCD28 (10 µg/mL) for 3 days with polarizing cytokine and antibodies to promote the differentiation of Th9 (10 µg/mLaIFN-g, 20 ng/mL mIL-4, 10ng/mL hIL-2, 5 ng/mL hTGF-β) for 3 days. After 3 days, cells were washed and cultured without aCD3 or aCD28 but with polarizing cytokines and antibodies, and with 10 ng/mL hIL-2, in fresh media. Media, cytokines, and antibodies were changed every 2 days. Cells were collected 5 days later (on d8) for analysis
Growth protocol Complete IMDM (10% FBS, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 55 μM 2-Mercaptoethanol
Extracted molecule genomic DNA
Extraction protocol ChIP-seq sample preparation was performed with Covaris kit (PN 520154). Naïve T cells were cultured in vitro under Th9 promoting conditions and treated with chemical cross-linking (1% formaldehyde) on days 0, 3, 4, and 8 (murine) or days 0, 5, 8, and 15 (human). At least 0.5 million cells were used for each immunoprecipitation. After chemical chromatin cross-linking (1% formaldehyde), cells were washed and frozen at -80C. Cells were resuspended in Lysis Buffer B (Covaris) with protease inhibitors, and DNA fragmentation was performed on a Covaris Sonicator (ME220) for 8 min to an average length of 200-700 bp. After sonication, cells were immunoprecipitated with anti-H3K27Ac (ab4729; Abcam), anti-H3K4Me1 (ab8895; Abcam), and anti-H3K4Me3 (ab8580; Abcam). Genomic DNA (input) was prepared by treating aliquots of chromatin with proteinase K, and heated for de-cross linking, followed by column-based DNA purification (28106, Qiagen). An aliquot of chromatin underwent immunoprecipitation, after which decrosslinking was performed as above.
After recovering purified DNA, 5ng or more of DNA was used to generate libraries using the NEBNext Ultra Directional II DNA sequencing kit per the manufacturer’s instructions.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
 
Data processing Basecalls were performed using bcl2fastq v2.20.0.422
Reads of 50 bases were processed using the ccbr pipeliner tool (https://ccbr.github.io/pipeliner-docs/): adapter sequences were trimmed with cutadapt 1.18
Data were mapped to the mouse genome mm10 or human genome GRCh38 (hg38)  using BWAmem 0.7.17. Uniquely mapped and nonredundant reads were generated using Picard 2.17.11 and used for downstream analysis.
The aligned files were converted to bam format using SAMtools 1.6, and bigwig files were created using deepTools 3.0.1.
For downstream analysis, peaks were called using MACS v2.1.1 using a FDR 0.01 and with the input sample for background correction and using the --broad setting
Assembly: mm10 (mouse), hg38 (human)
Supplementary files format and content: bedfile, broadPeak
 
Submission date Jan 14, 2023
Last update date Apr 28, 2023
Contact name Daniella Schwartz
E-mail(s) Daniella.Schwartz@pitt.edu
Organization name University of Pittsburgh
Street address 200 Lothrop St
City Pittsburgh
State/province PA
ZIP/Postal code 15216
Country USA
 
Platform ID GPL24247
Series (2)
GSE222908 ChIP-seq of in vitro differentiated naïve and Th9 cells
GSE222910 ATAC-seq, ChIP-seq and RNA-seq of in vitro differentiated naïve, Th1, and Th9 cells
Relations
BioSample SAMN32740038
SRA SRX19035355

Supplementary file Size Download File type/resource
GSM6935085_M1_D8_K27Ac_S14_peaks.bed.gz 1.1 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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