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Sample GSM5935359 Query DataSets for GSM5935359
Status Public on Apr 19, 2022
Title GFP-_BM
Sample type SRA
 
Source name bone marrow progenitor cells
Organism Mus musculus
Characteristics cell type: GFP-NFIL3 negative BM
strain: C57BL/6
chip or cut&run antibody: NFIL3 (Santa Cruz Biotechnology, sc-9550X, Lot# G0815)
Treatment protocol Nfil3GFP/GFP mice were injected once daily, subcutaneously, with 10 mg recombinant Flt-3L-Ig (BE0098; Bio X Cell) for three consecutive days to expand GFP-NFIL3+ DC progenitor cell numbers.
Growth protocol BM cells were isolated from Nfil3GFP/GFP mice and CD3-, CD19-, B220-, CD105-, TER-119-, Ly-6G-, CD4-, CD8b-, CD11b-, Ly-6C- and MHCII-expressing lineage-committed cells were depleted. GFP-NFIL3 negative BM and GFP-NFIL3 positive BM cells were sort purified.
Extracted molecule genomic DNA
Extraction protocol 0.7 million fresh sort purified cells were adsorbed to activated Concanavalin A (ConA) beads, permeabilized with 0.01% digitonin, and incubated with 0.5 mg goat-anti-NFIL3 antibody 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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Data processing Library strategy: CUT&RUN
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 Mar 06, 2022
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 SAMN26490302
SRA SRX14389436

Supplementary file Size Download File type/resource
GSM5935359_GFP-_BM.tags.ucsc.bedGraph.gz 39.4 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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