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Sample GSM5683645 Query DataSets for GSM5683645
Status Public on Apr 19, 2022
Title D1+2+3_C/EBPa
Sample type SRA
 
Source name bone marrow progenitor cells
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)
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.
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.
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
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
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.
 
Submission date Nov 09, 2021
Last update date Apr 19, 2022
Contact name Tiantian Liu
E-mail(s) ltt0321@gmail.com
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
Relations
BioSample SAMN23009387
SRA SRX13084917

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record
Processed data provided as supplementary file

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