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Status |
Public on Apr 19, 2022 |
Title |
D1+2+3_C/EBPa |
Sample type |
SRA |
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Source name |
bone marrow progenitor cells
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Organism |
Mus musculus |
Characteristics |
cell type: Hoxb8 hematopoietic progenitor cell line stably express C/EBPa strain: C57BL/6 chip or cut&run antibody: C/EBPa (Cell Signaling Technology, 8178S, Lot3)
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Treatment protocol |
The Hoxb8 cell lines stably expressing Flag-NFIL3 or C/EBPa were generated by retrovirally transducing with MSCV-Flag-NFIL3-IRES-GFP or MSCV-C/EBPa-T2A-Thy1.1 vectors and sorting for expression of GFP or Thy1.1.
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Growth protocol |
BM cells were isolated from 6 week old WT or D1+2+3 mice and CD3-, CD19-, CD105-, TER-119-, Ly-6G- and B220-expressing lineage-committed cells were depleted. The lineage– BM cells were cultured in complete IMDM supplemented with 50 ng/ml SCF (Peprotech), 25 ng/ml IL-3 (Peprotech), 25 ng/ml IL-6 (Peprotech), and 5% Flt3L-conditioned medium for 2 days and then retrovirally transduced with MSCV-Neo-ER-Hoxb8. Three days after infection, 1 mM β-estradiol (Sigma Aldrich) and 1 mg/ml G418 were added to select and maintain growth of ER-Hoxb8-transduced cells.
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Extracted molecule |
genomic DNA |
Extraction protocol |
0.5 million fresh cells were adsorbed to activated Concanavalin A (ConA) beads, permeabilized with 0.01% digitonin, and incubated with 0.5 mg indicated antibodies overnight at 4 °C. After antibody binding, the targete-DNA complex is cleaved by the Protein A/Protein G-Micrococcal Nuclease (pAG-MNase) for 2 hours at 4 °C. 0.05ng E. coli Spike-in DNA was added before releasing CUT&RUN fragments at 37°C for 10 min. The CUT&RUN enriched DNA is purified from the supernatant. Libraries were prepared with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and cleaned-up with AMPureXP. OTHER: CUT&RUN
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
The adaptor sequences were removed by Trimmomatic program: java -jar trimmomatic-0.39.jar PE -threads 30 -phred33 WT_CEBPa_R1.fastq.gz WT_CEBPa_R2.fastq.gz -baseout WT_CEBPa.fastq.gz ILLUMINACLIP:Truseq3.PE.fa:2:15:4:4:true LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:25 The trimmed reads were aligned and mapped to the mouse reference genome (GRCm38/mm10) by Bowtie2 software version 2.2.5: bowtie2 -p 30 --dovetail --phred33 -x mm10 -1 WT_CEBPa_1P.fastq.gz -2 WT_CEBPa_2P.fastq.gz > WT_CEBPa.sam Duplicated reads are discarded using 'make tag directory' of Homer software package (version 4.9) with the parameter -tbp 1: makeTagDirectory WT_CEBPa.tags WT_CEBPa.sam -fragLength given -tbp 1 Data were visualized with the 'makeUCSCfile' of Homer: makeUCSCfile WT_CEBPa.tags -o auto -fragLength given -tbp 1 -fsize 5e7 Peak calling was performed with Homer software package: findPeaks WT_CEBPa -style factor -size 200 -o WT_CEBPa.txt -i negative_control -poisson 1e-10 Genome_build: mm10 Supplementary_files_format_and_content: bedGraph files were generated with Homer software package using the "makeUCSCfile" command.
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Submission date |
Nov 09, 2021 |
Last update date |
Apr 19, 2022 |
Contact name |
Tiantian Liu |
E-mail(s) |
ltt0321@gmail.com
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Organization name |
Washington University in St. Louis
|
Department |
Pathology and Immunology
|
Lab |
Dr. Kenneth Murphy
|
Street address |
660 S. Euclid Ave.
|
City |
Saint Louis |
State/province |
MO |
ZIP/Postal code |
63110 |
Country |
USA |
|
|
Platform ID |
GPL24247 |
Series (2) |
GSE188482 |
Ablation of cDC2 specification by triple mutations in the Zeb2 enhancer [CUT&RUN] |
GSE188579 |
Ablation of cDC2 development by triple mutations within the Zeb2 enhancer |
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Relations |
BioSample |
SAMN23009387 |
SRA |
SRX13084917 |