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| Status |
Public on Mar 01, 2023 |
| Title |
ChIP_IgG_KO_LPS8h_S20 |
| Sample type |
SRA |
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| Source name |
BMDM
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| Organism |
Mus musculus |
| Characteristics |
cell line: BMDM
cell type: bone marrow-derived macrophages
chip antibody: IgG (Cell Signaling 2729)
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| Treatment protocol |
Cells were treated at time points with 100 ng/mL LPS.
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| Growth protocol |
BMDM were generated from bone marrow prepared for in vitro differentiation for 5 days in complete Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, # 22400089) supplemented with 10% (v/v) fetal bovine serum, 20 ng/mL recombinant murine MCSF (Preprotech, # 315-02), 100 U/mL Penicilin-Streptomycin and 2 mM L-Glutamine, at 37 oC and 5% CO2 atmosphere.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~1x107 BMDM were briefly fixed with 1% methanol-free formaldehyde solution (Thermo Scientific, # 28908) for 1 min at RT. Then, reaction was quenched with addition of 125 mM glycine for 3 min. Cells were harvested in ice-cold PBS (pH 7.4) and centrifuged at 300 x g for 5 min at 4 oC. Chromatin isolation and immunoprecipitation were carried out using CUTANATM ChIC/CUT&RUN kit (EpiCypher #141048). One hundred thousand cells were resuspended in Wash Buffer (Pre-Wash Buffer supplemented with 1X protease inhibitor cocktail, 460 mM Spermidine, 1% Triton X-100, 0.045% SDS), and incubated with activated ConA beads for 10 min at RT. Then, cells were permeabilized with 0.01% Digitonin in Wash Buffer, and incubated with 0.5 g target antibody at 4 oC overnight with agitation. The antibodies used were anti-p65 (Cell Signaling, # 8242), anti-SOCS1 (Cell Signaling, # 3950), or Normal Rabbit IgG (Cell Signaling, # 2729). Cells were washed twice with cold Wash Buffer supplemented with Digitonin, and chromatin regions around target-DNA complex were digested with pAG-MNase for 10 min at RT, followed by addition of 2 mM CaCl2, and incubation with agitation for 2 h at 4 oC. Digestion was stopped with addition of Stop Buffer, and E. coli Spike-in DNA was added to each sample for normalization. Target-DNA complex fragments were released by heat shock at 37 oC for 10 min, followed by magnetic separation of cells. Cross linking was reversed by digestion of proteins in the supernatant with 20 g Proteinase K at 55 oC overnight. Chromatin fragments were purified by standard protocol using DNA Cleanup Columns provided in the kit. IL).
Sample quality, library complexity, and alignment statistics were checked using an established pipeline at the NUSeq Core Facility at Northwestern University (Chicago,
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Reads were trimmed to remove Illumina adapters from the 3’ ends using cutadapt
Trimmed reads were aligned to the Mus musculus genome (mm10) using Bowtie2 (version 2.4.1) with default parameters while only reads that mapped uniquely to the genome were used in subsequent analysis.
ChIP-seq analysis was performed largely using Hypergeometric Optimization of Motif EnRichment (HOMER, version 4.11) (Duttke et al., 2019). HOMER was used to call peaks, identify loci of differential binding, generate UCSC read density tracks, annotate peaks, and detect motif enrichments.
Heatmaps of TSS enrichment of were generated using deepTools2
Assembly: mm10
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| Submission date |
Feb 22, 2023 |
| Last update date |
Mar 03, 2023 |
| Contact name |
Brian Wray |
| E-mail(s) |
brianwray26@gmail.com
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| Organization name |
Northwestern University
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| Department |
Feinberg School of Medicine
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| Street address |
633 Clark Street
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| City |
Evanston |
| State/province |
IL |
| ZIP/Postal code |
60208 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE225833 |
SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes (ChIP-Seq). |
| GSE225835 |
SOCS1 regulates a subset of NFκB-target genes through direct chromatin binding and defines macrophage functional phenotypes |
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| Relations |
| BioSample |
SAMN33417480 |
| SRA |
SRX19473914 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7057425_ChIP_IgG_KO_LPS8h.bw |
221.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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