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Sample GSM8016302 Query DataSets for GSM8016302
Status Public on Mar 05, 2024
Title BMMC, PBS, H3K4me1, rep2
Sample type SRA
 
Source name Cultured primary bone marrow monocytes
Organism Mus musculus
Characteristics cell type: Cultured primary bone marrow monocytes
strain: C57Bl/6J
age: 8-weeks-old
Sex: Female
treatment: PBS
antibody: H3K4me1 (EpiCypher #13-0057)
Treatment protocol BMMCs were cultured for 5 days under continuous high-dose 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) stimulation or PBS control conditions
Growth protocol In vitro bone marrow monocyte (BMMC) LPS exhaustion experiments were performed as previously described (Pradhan et al., 2021, Front. Immunol.). Briefly, bone marrow cells were harvested from 6- to 8-week-old C57BL/6 WT female mice, seeded at a density of 3x105 cells/cm2, and cultured in complete RPMI 1640 media (10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine) supplemented with 10 ng/mL M-CSF (PeproTech). Cells were cultured for 5 days at 37˚C in a humidified 5% CO2 atmosphere under continuous high-dose 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) stimulation or PBS control conditions, with fresh media changes at days 2 and 4 of culture.
Extracted molecule genomic DNA
Extraction protocol Cultured BMMCs were harvested by manual disruption and immediately processed for CUT&RUN using a CUTANA ChIC/CUT&RUN Kit (EpiCypher) according to the manufacturer’s protocol. Briefly, live cells were permeabilized in 0.01% digitonin in the presence of ConA beads. K-MetStat Panel spike-in was added to each reaction, and samples were incubated overnight at 4˚C in the presence of H3K4me1 (EpiCypher #13-0057), H3K4me3 (EpiCypher #13-0041), or IgG negative control (EpiCypher #13-0042) antibodies. Following PAG-MNase digestion and chromatin release, 0.5ng of E. coli spike-in DNA were added to each reaction, and DNA samples were purified using CUTANA kit reagents.
Purified DNA was quantified using a Qubit dsDNA HS Assay Kit (Invitrogen), and 1-5 ng of purified DNA were used as input for CUTANA CUT&RUN Library Prep Kit (EpiCypher) according to the manufacturer’s protocol. Library fragment sizes were analyzed using Tapestation DNA ScreenTape analysis (Agilent) at the Fralin Life Sciences Institute at Virginia Tech Genomics Sequencing Center.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model NextSeq 1000
 
Data processing Paired-end CUT&RUN reads were mapped to the mm10 and E. coli (MG1655) genomes using bowtie2 (2.5.2; parameters: --local –very-sensitive –no-mixed –no-discordant –dovetail)
Low quality mapped reads (-q 30) and PCR duplicates were removed using samtools, and peak calling was performed using SEACR (parameters: norm, stringent)
CUT&RUN coverage was determined using deepTools (3.5.2; function: bamCompare), with coverage values normalized to E. coli spike-in controls
bigwig files were prepared using bedGraphToBigWig (UCSC)
Assembly: mm10
Supplementary files format and content: biwgwig, bed (SEACR formatted: <chr> <start> <end> <total signal> <max signal> <max signal region>)
Library strategy: CUT&RUN
 
Submission date Jan 13, 2024
Last update date Mar 05, 2024
Contact name Blake Alexander Caldwell
E-mail(s) blakeac@vt.edu
Phone 8655997842
Organization name Virginia Tech
Department Biological Sciences
Lab Li Lab
Street address 970 Washington Street SW
City Blacksburg
State/province Virginia
ZIP/Postal code 24061
Country USA
 
Platform ID GPL32159
Series (2)
GSE253201 Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis [CUT&RUN]
GSE253202 Altered DNA methylation underlies monocyte dysregulation and immune exhaustion memory in sepsis
Relations
BioSample SAMN39432661
SRA SRX23206505

Supplementary file Size Download File type/resource
GSM8016302_PBS-H3K4me1-2.bw 49.5 Mb (ftp)(http) BW
GSM8016302_PBS-H3K4me1-2.stringent.bed.gz 1.1 Mb (ftp)(http) BED
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Raw data are available in SRA

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