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Status |
Public on Apr 19, 2022 |
Title |
Hoxb8_NFIL3_IP |
Sample type |
SRA |
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Source name |
bone marrow progenitor cells
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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)
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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.
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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.
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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.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2500 |
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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.
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Submission date |
May 08, 2021 |
Last update date |
Apr 27, 2022 |
Contact name |
Tiantian Liu |
E-mail(s) |
ltt0321@gmail.com
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Organization name |
Washington University in St. Louis
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Department |
Pathology and Immunology
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Lab |
Dr. Kenneth Murphy
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Street address |
660 S. Euclid Ave.
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City |
Saint Louis |
State/province |
MO |
ZIP/Postal code |
63110 |
Country |
USA |
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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 |
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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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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