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| Status |
Public on Jan 16, 2024 |
| Title |
BMDM_LA_0h, scATAC-seq |
| Sample type |
SRA |
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| Source name |
primary macrophages
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| Organism |
Mus musculus |
| Characteristics |
tissue: Bone Marrow
cell type: mouse bone marrow derived macrophages (BMDM)
genotype: WT
treatment: untreated
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| Treatment protocol |
BMDM on day 6 was stimulated with 100ng/ ml final concentration of Lipid A (Sigma Aldrich, Cat# L6895-1MG)
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| Growth protocol |
Bone marrow precursors were grown in M-CSF conditioned media and FBS for 6 days prior to stimulation as described in Purbey et al., 2017 (PMID:28930658).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The transposition reaction was prepared by mixing about 12000 nuclei (to finally recover about 8000 nuclei) prepared from unstimulated and stimulated samples with ATAC Buffer and Enzyme and incubated for 30 min at 37 °C as per 10X scATAC-seq guide (CG000496 Rev B). Nuclei preparation: Both untreated and stimulated cells were washed twice with DPBS ( Thermo Fisher, Cat # 14190144) and incubated with 1ml Accutase (Sigma Aldrich, Cat # A6968) for 8 minutes at 37 0C and then diluted with additional 2 ml of DPBS + 0.04% BSA. Single cell suspension was prepared by gently dislodging by splashing and pipetting the DPBS (3-4 times). The resuspended cells were pelleted in a 5 ml Eppendorf tube (300g for 5 min at 4 0C) and then washed with 1ml of DPBS + 0.04% BSA. The pellet was flicked resuspended in 50ul DPBS + 0.04% BSA and then lysed with 200 ul nuclei lysis buffer (10 mM Tris-HCl “pH 7.4”, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20, 0.1% NP-40 substitute, 0.01% digitonin and 1% bovine serum albumin “BSA; Thermo Fisher, Cat# AM2616”) by pipetting 8 times and vortexing for 10 sec and by incubating for 8 minutes on ice (with gentle flicking of the cells every 2 mins). The pellet was washed with 1 ml of chilled wash buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20 and 1% BSA) by pipetting 5-6 times. Nuclei were pelleted at 500gfor 5 min at 4 °C, and the supernatant was removed carefully without disrupting the nuclei pellet. Assuming, about 50% loss during the procedure, nuclei were resuspended into 60 ul of chilled 1X Nuclei Buffer (10x Genomics).
libraries were constructed Chromium Next GEM Single Cell ATAC Reagent kits v2 (10X Genomics) according to manufactuer's instruction.
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| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
scATAC-seq libraries were sequenced paired-end at 100 cycle in an Illumina NovaSeq 6000 S1 platform.
FASTQ files were generated from binary base call files using Cell Ranger ATAC v.2.0.0 mkfastq command
FASTQ files were used to aligned to the mm10 mouse reference genome using Cell Ranger ATAC (v2.0.0 ‘cellranger-atac count’) with default settings.
Used "cellranger-atac count" for Read alignment, barcode counting and peak calling
Final analysis were perfomed in R, Signac, Seurat and ggplot2 softwares.
Assembly: mm10
Supplementary files format and content: "filtered_peak_bc_matrix.h5" file contains the filtered peak-barcode matrix from produced by Cell Ranger ATAC V2 pipeline, which is suitable for analysis with Signac in R.
Supplementary files format and content: "fragments.tsv.gz" file contains information about each DNA fragment . This includes the genomic coordinates of each fragment and the corresponding cell barcode.
Supplementary files format and content: "fragments.tsv.gz.tbi" file contains index information and allows for quick access to the compressed scATAC-seq fragment data in "fragments.tsv.gz" files during downstream analysis.
Supplementary files format and content: "singlecell.csv" file contains a matrix of chromatin accessibility across cells, with barcodes for cell identification, genomic loci of open chromatin, quality metrics and metadata for each cell, which is useful for down stream analysis in R using Signac.
Supplementary files format and content: "web_summary.html" file is HTML-formatted report that details key metrics from scATAC-seq data analysis by Cell Ranger V2. This includes information about sequencing depth, peak detection, and library quality, which can be viewed in a web browser for easy assessment
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| Submission date |
Nov 27, 2023 |
| Last update date |
Jan 16, 2024 |
| Contact name |
Stephen Smale |
| E-mail(s) |
smale.geo.uploads@gmail.com
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| Organization name |
University of California Los Angeles
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| Department |
MIMG
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| Lab |
Smale Lab
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| Street address |
6-730 MRL 675 Charles E. Young Drive South
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| City |
Los Angeles |
| State/province |
Ca |
| ZIP/Postal code |
90024 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE234914 |
Toll-Like Receptor-Induced Nucleosome Remodeling Achieved by Broadly Acting NF-kB in Collaboration with Transcription Factors Conferring Selectivity |
| GSE248713 |
NF-kB Broadly Orchestrates Nucleosome Remodeling during the Primary Response to Toll-Like Receptor 4 Signaling [scATAC-seq] |
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| Relations |
| BioSample |
SAMN38455226 |
| SRA |
SRX22656963 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7919028_1B_BMDM_LA_0h_filtered_peak_bc_matrix.h5 |
127.3 Mb |
(ftp)(http) |
H5 |
| GSM7919028_1C_BMDM_LA_0h_fragments.tsv.gz |
2.0 Gb |
(ftp)(http) |
TSV |
| GSM7919028_1D_BMDM_LA_0h_fragments.tsv.gz.tbi.gz |
970.5 Kb |
(ftp)(http) |
TBI |
| GSM7919028_1E_BMDM_LA_0h_singlecell.csv.gz |
5.6 Mb |
(ftp)(http) |
CSV |
| GSM7919028_1F_BMDM_LA_0h_web_summary.html.gz |
1002.2 Kb |
(ftp)(http) |
HTML |
SRA Run Selector |
| Raw data are available in SRA |
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