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GEO help: Mouse over screen elements for information. |
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Status |
Public on Apr 06, 2020 |
Title |
Gfi1 wild-type 10x scATAC-Seq replicate 2 |
Sample type |
SRA |
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Source name |
Bone marrow
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 cell type: Bone marrow derived mixed-gating strategy genotype: Wild-type cdna synthesis: 10x Genomics single cell 3’ v2 assay protocol library prep: Chromium Single Cell ATAC Reagent Kits User Guide (v1 Chemistry) sequencing parameters: 493 million reads
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Extracted molecule |
genomic DNA |
Extraction protocol |
Bone marrow was extracted by crushing hindlimbs isolated from mice immediately following euthanasia. The following bone marrow cell populations were FACS-sorted: proNeu-2 (Live Modified Lin- CD34+ CD16/32+ CD117+ Vcam1+), preNeu-1 (Live Modified Lin- CD34+ CD16/32+ CD117+ Ly6c+ CD11b- CD115- Vcam1+), preNeu-2 & preNeu-3 (Live Modified Lin- CD34+ CD16/32+ CD117+ Ly6c+ CD11b+ CD115- Ly6g dim-mid),), immNeu (Live Modified Lin- CD34+ CD16/32+ CD117+ Ly6c+ CD11b+ CD115- Ly6g high), and Modified GMP (Live Modified Lin- CD34+ CD16/32+). See https://support.10xgenomics.com/single-cell-atac for additional details. Cells were processed according to ‘Demonstrated Protocol – Nuclei Isolation for Single Cell ATAC Sequencing, CG000169 Rev B). Isolated nuclei (12,000-20,000) were captured with a Chromium Controller Instrument (10x Genomics) using the Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit according to the manufacturer’s instructions. Nuclei from two independent Gfi1-wild-type animals were loaded onto two separate ports of the same 10x Genomics Chromium chip and sequenced to a combined target depth of ~1 billion reads. Nuclei from a single Gfi1-R412X-R412X animal was laoded onto a port on the 10x Genomics Chromium chip.
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Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Male and Female mixed gating strategy DNA
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Data processing |
The fastq files for each sample generated from scATAC-Seq sequencing were processed in mm10 using the cellranger-atac count program (version 1.0.1)(10X Genomics) [https://www.ncbi.nlm.nih.gov/pubmed/28091601] (“cellranger-atac count –id – reference –fastqs”). The two replicate Gfi1+/+ mouse scATAC-Seq datasets produced 14,766 and 15,186 filtered nuclei barcodes with 11,220 and 18,402 median fragments per nuclei, respectively. The one Gfi1R412X/R412X mouse scATAC-Seq dataset produced 15,767 filtered nuclei barcodes and 8,305 median fragments per nuclei. The resulting “filtered peak bc matrix” was used as input to downstream analysis of identifying cell clusters with Seurat (version 3.0.2), following downsampling of Gfi1+/+ capture reads to that of the Gfi1R412X/R412X (samtools) [Stuart, T., Butler, A., Hoffman, P., Hafemeister, C., Papalexi, E., Mauck III, W. M., ... & Satija, R. (2019). Comprehensive Integration of Single-Cell Data. Cell.]. Following cicero analysis and filtering (default parameters), 18,457 Gfi1+/+ and 7,692 Gfi1R412X/R412X barcodes were retained. To perform label transfer in Seurat, mapping scATAC-Seq to scRNA-Seq expression values, a gene activity matrix was produced from the combined Gfi1+/+ replicates, as well as the Gfi1R412X/R412X sample with the program Cicero (version 1.0.15) as previously described [Pliner, H. A., Packer, J. S., McFaline-Figueroa, J. L., Cusanovich, D. A., Daza, R. M., Aghamirzaie, D., ... & Adey, A. C. (2018). Cicero predicts cis-regulatory DNA Interactions from single-cell chromatin accessibility data. Molecular cell, 71(5), 858-871.]. The first 30 principal components were used for “FindNeighbors” and a resolution of “1.2” was used for “FindClusters”. Transfer anchors were found using the top 5,000 variable features and cell type predictions were made with the highest scoring prediction used as the cell label for downstream analysis, constituting labelled clusters from Seurat. The .bam alignment file generated by the cellranger-atac count program was then split into a separate file for each cluster based on the “CB” barcode tag associated with each read, which was used as input to IGV to display coverage for each cell state, normalized by the total number of reads. Predicted nuclei clusters from the default cellranger-atac graphclust software were also compared to the Seurat cluster predictions at the indicated resolutions (Extended Data Fig. 12b). Additionally, the SNAP-ATAC workflow was run in parallel on these data, using the default options to compare to the Seurat clusters [https://www.biorxiv.org/content/10.1101/615179v2. Both cellranger-atac graphclust as well as SNAP-ATAC provided unbiased analyses to cluster cells. These were compared to the label transfer program of Seurat by calculating the percentage of cells shared between clusters. To test for the accessibility of regions at likely Gfi1 binding locations, these files were used to compute coverage across the +/- 500 bp region for each peak found in the ChIP-Seq data analysis, which was then normalized by the total number of cells associated to each cluster in the separate .bam files. Genome_build: mm10 Supplementary_files_format_and_content: Tab-delimited text file and HDF5 Supplementary_files_format_and_content: clusters.Gfi1-WT-rep-3531.txt: Cell Clusters: Seurat 3 assigned cluster labels for Gfi1-WT nuclei (replicate 1) Supplementary_files_format_and_content: clusters.Gfi1-WT-rep-3534.txt: Cell Clusters: Seurat 3 assigned cluster labels for Gfi1-WT nuclei (replicate 2) Supplementary_files_format_and_content: clusters.Gfi1-R412X-R412X.txt: Cell Clusters: Seurat 3 assigned cluster labels for Gfi1-R412X-R412X nuclei Supplementary_files_format_and_content: Gfi1-WT-rep-3531-filtered_tf_bc_matrix.h5: DNA Accessiblility TF motifs: TF-barcode matrix for Gfi1-WT nuclei (replicate 1) Supplementary_files_format_and_content: Gfi1-WT-rep-3534-filtered_tf_bc_matrix.h5: DNA Accessiblility TF motifs: TF-barcode matrix for Gfi1-WT nuclei (replicate 2) Supplementary_files_format_and_content: Gfi1-R412X-R412X-filtered_tf_bc_matrix.h5: DNA Accessiblility TF motifs: TF-barcode matrix for Gfi1-R412X-R412X nuclei Supplementary_files_format_and_content: Gfi1-WT-rep-3531-filtered_peak_bc_matrix.h5: DNA Accessiblility peaks: UMI counts for Gfi1-WT nuclei (replicate 1) Supplementary_files_format_and_content: Gfi1-WT-rep-3534-filtered_peak_bc_matrix.h5: DNA Accessiblility peaks: UMI counts for Gfi1-WT nuclei (replicate 2) Supplementary_files_format_and_content: Gfi1-R412X-R412X-filtered_peak_bc_matrix.h5: DNA Accessiblility peaks: UMI counts for Gfi1-R412X-R412X nuclei
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Submission date |
Dec 19, 2019 |
Last update date |
Apr 08, 2020 |
Contact name |
H. Leighton Grimes |
E-mail(s) |
Lee.Grimes@cchmc.org
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Phone |
513-636-6089
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Organization name |
Cincinnati Childrens Hospital Medical Center
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Department |
Immunobiology
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Lab |
Grimes
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Street address |
3333 Burnet Ave. MLC 7038
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City |
Cincinnati |
State/province |
OH |
ZIP/Postal code |
45229 |
Country |
USA |
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Platform ID |
GPL24247 |
Series (2) |
GSE120409 |
A neutropenia-associated transcription factor mutation differentially impacts target genes in the cell-states traversed during granulocyte specification and commitment |
GSE142358 |
Mouse models of neutropenia reveal progenitor-stage-specific defects [scATAC-Seq] |
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Relations |
BioSample |
SAMN13635147 |
SRA |
SRX7416563 |
Supplementary file |
Size |
Download |
File type/resource |
GSM4226309_Gfi1-WT-rep-3534-filtered_peak_bc_matrix.h5 |
150.6 Mb |
(ftp)(http) |
H5 |
GSM4226309_Gfi1-WT-rep-3534-filtered_tf_bc_matrix.h5 |
9.0 Mb |
(ftp)(http) |
H5 |
GSM4226309_clusters.Gfi1-WT-rep-3534.txt.gz |
44.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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