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| Status |
Public on Nov 25, 2020 |
| Title |
BMMCs, Gata2_WT_ST_Input |
| Sample type |
SRA |
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| Source name |
BMMCs
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 * 129
cell type: Bone-marrow derived mast cells
chip antibody: none
genotype: wild type
treatment: IgE anti_TNP and TNP-BSA
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| Treatment protocol |
(Stimulation) BMMCs were sensitized with 1 ug/mL IgE anti-2, 4, 6-Trinitrophenyl (TNP) antibody (100 ng/mL) (IGEL 2a, ATCC, Manassas, VA). Twenty-four hours later, the cells were challenged with 100 ng/mL TNP-BSA (T-5050-100, LGC Biosearch Technologies, Novato, CA) for 2 additional hours before the cells were collected for further analysis.
(Gata2 deletion) BMMCs cultured from bone marrow cells of Gata2f/fRosaYfp/YfpTgCreErt2hemi mice or Gata2+/+RosaYfp/YfpTgCreErt2hemi mice were treated with 25 nM 4-hydroxytamoxifen (4-OHT; Calbiochem, Billerica, MA) for five days as described previously (Li et al., 2018; Li et al., 2015). The expression of FceR1a (PE-CY7–conjugated anti-FcεRIa (MAR-1), Biolegend, 134317) and c-Kit (allophycocyanin-CY7–conjugated anti–c-Kit (2B8), Biolegend, 105825) on mast cells were analyzed by Flow cytometry. The YFP+ cells were FACS-sorted and the deletion of the Gata2 gene was determined to be near 100%. The sorted cells were then used for the next-generation sequencing analysis.
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| Growth protocol |
Bone marrow-derived mast cells (BMMCs) were cultured from bone marrow cells of Balb/c mice in Iscove’s DMEM (10016CV, CorningTM cellgroTM, Manassas, VA) plus 10% FBS,100 units/mL penicillin, 100 ug/mL streptomycin, and 2 mM beta-mercaptoethanol in the presence of 20 ng/mL IL-3 for four weeks. Over 99% of BMMCs were mast cells as determined by FACS analysis (FcεRIa+ c-Kit+).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 1x10e7 BMMCs that were not treated or treated with IgE receptor crosslinking were fixed in complete medium with 1% Formaldehyde (Thermo Scientific, 28908). The fixation was quenched by adding glycine and then washed with ice-cold PBS. The cells were resuspended in lysis buffer with protease inhibitor cocktail (Sigma, SRE0055-1BO) and sonicated using Covaris S220 Focused-ultrasonicator (Covaris, Woburn, MA). The lysate was pre-cleared by incubating with protein A agarose/salmon sperm DNA slurry (Millipore, Cat# 16-157) and the supernatant was incubated with the anti-H3K4me1 antibody (ab8895, Abcam, Cambridge, MA) or anti-H3K27ac antibody (ab4729, Abcam) at 4 °C overnight. Then, the samples were incubated with salmon sperm DNA/protein A beads at 4 °C for one hour. The beads were washed using low salt immune complex wash buffer, high salt immune complex wash buffer once, LiCl immune complex wash buffer once and TE buffer twice. The beads were eluted with elution buffer. The crosslinking of eluted immunocomplexes was reversed by incubating with NaCl and RNase A (Roche, Cat# 11119915001) at 65 °C overnight, followed by incubation with Tris pH 6.5, EDTA pH 8.0, and Proteinase K at 55 °C for one hour. Finally, the recovered DNA was cleaned up using a QIAGEN QIAquick PCR purification kit (Qiagen, Valencia, CA).
ChIP-seq library was prepared using TruSeq ChIP Library Preparation Kit (IP-202-1024, Illumina, San Diego, CA) according to the manufacturer’s instructions. Briefly, 10 ng of ChIPed DNA was converted into blunt-ended fragments. A single adenosine nucleotide was added to the 3’ ends of the blunt-ended fragments before indexing adaptors were ligated to the adenylated 3' ends. The ligated product was purified, size-selected and PCR amplified according to the manufacturer’s instructions. The quality and quantity of the DNA library were assessed on an Agilent Technologies 2100 Bioanalyzer. The paired-ended sequencing was performed on an Illumina NovaSEQ6000 platform.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The adaptor sequences in raw reads (average 24.0 million reads, two biological replicates for each group) were trimmed using Trimmomatic (version 0.33) (Bolger et al., 2014) and the quality of trimmed sequence data was analyzed by FastQC (version 0.11.9).
The trimmed reads were aligned to the mm10 reference genome using Bowtie2 (version 2.3.5.1) (Langmead and Salzberg, 2012).
Peak calling was performed using MACS2 (version 2.1.2) with default parameters (Zhang et al., 2008).
Genome_build: mm10
Supplementary_files_format_and_content: Tdf and bedgraph files for each sample
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| Submission date |
Feb 19, 2020 |
| Last update date |
Nov 25, 2020 |
| Contact name |
Hua Huang |
| E-mail(s) |
HuangH@njhealth.org
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| Organization name |
National Jewish Health
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| Department |
Biomedical Research
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| Street address |
1400 Jackson St
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| City |
Denver |
| State/province |
CO |
| ZIP/Postal code |
80206 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE145544 |
Transcription Factor GATA2 Promotes Chromatin Remodeling at the Super-Enhancers of the Key Mast Cell Identity Genes and Primes Enhancers to Respond to Antigenic Stimulation [ChIP-seq] |
| GSE145612 |
Transcription Factor GATA2 Promotes Chromatin Remodeling at the Super-Enhancers of the Key Mast Cell Identity Genes and Primes Enhancers to Respond to Antigenic Stimulation |
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| Relations |
| BioSample |
SAMN14136495 |
| SRA |
SRX7750193 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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