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| Status |
Public on Jun 27, 2023 |
| Title |
Mouse thymus stage 0 Va14 iNKT scATAC-seq |
| Sample type |
SRA |
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| Source name |
Thymus
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| Organism |
Mus musculus |
| Characteristics |
strain: V<alpha>14 transgenic (C57BL/6 background)
tissue: Thymus
cell type: Invariant natural killer T cells (iNKT) cells
age: 5 weeks
developmental stage: stage 0
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Murine thymi were harvested from 5 weeks old C57BL/6 mice or Vα14 transgenic mice. The thymus iNKT cells were enriched by positive selection using biotinylated antibodies against PE. Stage 1(CD24-CD44-NK1.1-), stage 2(CD24-CD44+NK1.1-) and stage 3(CD24-CD44+NK1.1+) were sorted from 5 weeks C57BL/6 mouse thymus, and Stage 0 iNKT cells (CD24+NK1.1-) were sorted from 5 weeks old Vα14 transgenic mouse thymus through BD FACS AriaII.
FACS-sorted NKT cells from B6 (stages 0, 1, 2, and 3) and Vα14 transgenic (stage 0) mouse thymi were used to generate Chromium Single Cell ATAC Libraries following the recommended protocol (10x Genomics, Pleasanton, CA). Briefly, after isolating, washing, and counting of nuclei suspensions, the recovered nuclei were combined with the ATAC buffer and enzyme to create a transposition mix in a tube and incubated at 37°C for 60 minutes. A master mix that include the barcoding reagent, reducing agent, and barcoding enzyme was then added to the same tube. This solution was loaded into the 10x Genomics Chromium Single Cell Controller instrument along with the vortexed single-cell ATAC gel beads and partition oil. The resulting GEMs were amplified in a Thermocycler (Veriti™ 96-Well Fast Thermal Cycler, Applied Biosynthesis; 72°C for 5 min, 98°C for 30 s, cycled 12x: 98°C for 10 s, 59°C for 30 s, and 72°C for 1 min). After cleaning up using the Recovery Agent, Dynabeads MyOne Silane, and SPRIselect Reagent, the resulting accessible DNA fragments underwent the barcoded and indexed sequencing library construction. These scATAC-seq libraries were sequenced on the Illumina NovaSeq 6000 using paired-end flow cells (50 bp Read 1, 8 bp i7 index, 16 bp i5 index, and 49 bp Read 2) by the University of Michigan DNA Sequencing Core facility following the manufacturer’s protocol.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Stage 0 iNKT cells from Va14 mouse thymus (sample 1)
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| Data processing |
Sequenced reads from scATAC-seq libraries were processed using the 10X Genomics Cell Ranger ATAC (v1.0.1).
Raw sequencing reads were demultiplexed and converted into FASTQ files usjng the cellranger-atac mkfastq pipeline. Then, the sequencing reads underwent the cellranger-atac count pipeline consisting of read filtering, alignment to the reference genome using STAR, barcode counting, identification of transposase cut sites and accessible chromatin peaks, cell calling, generation of count matrices for chromatin peaks and transcription factors.
Estimated number of cells captured per sample were between 2,536 - 11,015 with 11,401 - 16,343 median fragments per cell and 55,349 - 87,603 total peaks detected.
Each sample has 4 oligos with 3 FASTQ files of Read1 (R1), i5 barcode index (R2), Read2 (R3), and i7 sample index (I1). To generate the single-cell accessibility counts, we utilized the following command using the Cell Ranger ATAC in a Linux terminal: cellranger-atac count --id=<Sample_name> --sample=<Sample_name> --fastqs=<Path_to_fastq_files> --reference=<Path_to_Cell_Ranger_ATAC_compatible_genome_reference>
Genome_build: mm10
Supplementary_files_format_and_content: Cell Ranger ATAC outputs of barcodes, peak-barcode matrix, peaks, filtered peak barcode matrix in HDF5 format, barcoded and aligned fragments, fragment file index, per-barcode fragment counts and metrics.
TSV: Barcodes
MTX: Peak-barcode Matrix
BED: Peaks
H5: Filtered Peak Barcode Matrix in HDF5 Format
TSV.GZ: Barcoded and Aligned Fragments
TSV.GZ.TBI: Barcoded and Aligned Fragment File Index
CSV: Per-barcode Fragment Counts and Metrics
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| Submission date |
Dec 11, 2019 |
| Last update date |
Jun 28, 2023 |
| Contact name |
Indra Adrianto |
| E-mail(s) |
iadrian1@hfhs.org
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| Phone |
313-874-5999
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| Organization name |
Henry Ford Health System
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| Department |
Public Health Sciences
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| Street address |
1 Ford Place
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| City |
Detroit |
| State/province |
MI |
| ZIP/Postal code |
48202 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE141825 |
Single-cell ATAC-seq of mouse thymus iNKT cells |
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| Relations |
| BioSample |
SAMN13540754 |
| SRA |
SRX7347472 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4213749_Thy-St0-NKT-Va14_barcodes.tsv.gz |
30.3 Kb |
(ftp)(http) |
TSV |
| GSM4213749_Thy-St0-NKT-Va14_filtered_peak_bc_matrix.h5 |
91.6 Mb |
(ftp)(http) |
H5 |
| GSM4213749_Thy-St0-NKT-Va14_fragments.tsv.gz |
1.4 Gb |
(ftp)(http) |
TSV |
| GSM4213749_Thy-St0-NKT-Va14_fragments.tsv.gz.tbi.gz |
799.1 Kb |
(ftp)(http) |
TBI |
| GSM4213749_Thy-St0-NKT-Va14_matrix.mtx.gz |
183.7 Mb |
(ftp)(http) |
MTX |
| GSM4213749_Thy-St0-NKT-Va14_peaks.bed.gz |
702.1 Kb |
(ftp)(http) |
BED |
| GSM4213749_Thy-St0-NKT-Va14_singlecell.csv.gz |
5.9 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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