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Sample GSM5287384 Query DataSets for GSM5287384
Status Public on Apr 19, 2022
Title Hoxb8_NFIL3_IP
Sample type SRA
 
Source name bone marrow progenitor cells
Organism Mus musculus
Characteristics cell line: Hoxb8 hematopoietic progenitor cell line stably expressing Flag-NFIL3
strain: C57BL/6
chip antibody: NFIL3 (Santa Cruz Biotechnology, sc-9550X, Lot# G0815)
Treatment protocol The Hoxb8 cell line stably expressing Flag-NFIL3 was generated by retrovirally transducing with MSCV-Flag-NFIL3-IRES-GFP vector and sorting for expression of GFP.
Growth protocol BM cells were isolated from 6 week old 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-FLAG-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 Thirty million cells were crosslinked with 1% formaldehyde for 5 min, quenched with 1/20 volume of 2.5 M glycine and washed twice with PBS. To obtain nuclei, cell pellets were incubated in 1 mL Lysis buffer 1 (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40 and 0.25% Triton X-100) supplemented with protease inhibitors for 10 min, and 1 mL Lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) supplemented with protease inhibitors for 10 min. To shear chromatin, nuclei pellets were gently rinsed with Shearing buffer D3 (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, and 0.1% SDS) twice, and then sonicated at 4 °C in Shearing buffer D3 for 17 min, of 10% duty cycle, 75 Watts intensity peak incident power and 200 cycles per burst, with an ME220 focused-ultrasonicator (Covaris). A 5% fraction of lysate were kept for input, and the remaining lysate were immunoprecipitated overnight at 4 °C with Dynabeads Protein G (Invitrogen) that had been pre-incubated with 5 mg of goat anti-NFIL3 antibody (sc-9550X; Santa Cruz Biotechnology). Beads containing protein-DNA complexes were washed and DNA fragments were reverse-crosslinked and eluted.
Libraries were prepared with a ThruPLEX DNA-seq kit (Rubicon Genomics) and cleaned-up with AMPureXP.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
Data processing ChIP-seq reads were aligned and mapped to the mouse reference genome (GRCm38/mm10) by Bowtie software version 1.1.1: bowtie --sam --best -p 4 -t --verbose --trim5 5 -m 1 mm10 --chunkmbs 1000 Hoxb8_input.fastq > Hoxb8_input.sam; bowtie --sam --best -p 4 -t --verbose --trim5 5 -m 1 mm10 --chunkmbs 1000 Hoxb8_NFIL3_IP.fastq > Hoxb8_NFIL3_IP.sam
Duplicated reads are discarded using 'make tag directory' of Homer software package (version 4.9) with the parameter -tbp 1: makeTagDirectory Hoxb8_input.tags Hoxb8_input.sam -tbp 1; makeTagDirectory Hoxb8_NFIL3_IP.tags Hoxb8_NFIL3_IP.sam -tbp 1
Data were visualized with the 'makeUCSCfile' of Homer: makeUCSCfile Hoxb8_input.tags -o Hoxb8_input.bedgraph -fsize 5e7 -res 1; makeUCSCfile Hoxb8_NFIL3_IP.tags -o Hoxb8_NFIL3_IP.bedgraph -fsize 5e7 -res 1
Peak calling and de novo motif discovery were performed with Homer software package.
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph files were generated with Homer software package using the "makeUCSCfile" command.
 
Submission date May 08, 2021
Last update date Apr 27, 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 GPL17021
Series (2)
GSE174108 Ablation of cDC2 specification by triple mutations in the Zeb2 enhancer [ChIP-Seq]
GSE188579 Ablation of cDC2 development by triple mutations within the Zeb2 enhancer
Relations
BioSample SAMN19076306
SRA SRX10820639

Supplementary file Size Download File type/resource
GSM5287384_Hoxb8_NFIL3_IP.bedgraph.gz 31.6 Mb (ftp)(http) BEDGRAPH
GSM5287384_N3.bed.gz 104.2 Kb (ftp)(http) BED
GSM5287384_N3.txt.gz 189.7 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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