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| Status |
Public on Jun 27, 2024 |
| Title |
18-mo CD45- thymic endothelial cells - steady state [singe cell] |
| Sample type |
SRA |
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| Source name |
thymus
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| Organism |
Mus musculus |
| Characteristics |
tissue: thymus
strain: C57BL/6
cell type: CD45-PECAM1+ thymic stroma cells
genotype: wild type
age: 18-month old
treatment: untreated
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
All steps were performed at 4 C unless indicated. 10-15 thymi of 2-month-old or 18-month-old female C57BL/6 mice were excised and enzymatically digested. Briefly, thymi were mechanically dissociated into ca. 2 mm pieces. Tissue pieces were incubated with a digestion buffer (RPMI, 10% FCS, 62.5 um/mL liberase TM, 0.4 mg/ml DNase I) twice for 30 min at 37 C. Between incubation steps, supernatant containing dissociated cells was transferred to 50 mL conical tubes equipped with 100 um filter. Cells were pelleted by centrifugation at 400g for 5 min. Cell pellets were incubated with anti-mouse CD45 microbeads and CD45-positive cells were depleted from cell suspension using magnetic-associated cell sorting (MACS) on LS columns according to manufacturer’s protocol. Following red blood cell lysis using ACK buffer, the CD45-depleted cell fraction was incubated with an antibody cocktail for 15 min at 4 C and cells of interest were purified by fluorescent-associated cell sorting (FACS) on a BD Biosciences Aria II using a 100 um nozzle. Cells were sorted into tubes containing RPMI supplemented with 2% BSA. FACS-purified cells were spun down at 400g for 5 min and resuspended in PBS supplemented with 0.04 % BSA for generation of single-cell suspensions.
The single-cell RNA-seq of FACS-sorted cell suspensions was performed on Chromium instrument (10X genomics) following the user guide manual (CG00052 Rev E) and using Single Cell 3’ Reagent Kit (v2). The viability of cells prior to loading onto the encapsulation chip was 73-98%, as confirmed with 0.2% (w/v) Trypan Blue stain. Each sample, containing approximately 8000 cells, was encapsulated in microfluidic droplets at a final dilution of 66–70 cells/µl (a multiplet rate ~3.9%). Following reverse transcription step the emulsion droplets were broken, barcoded-cDNA purified with DynaBeads and amplified by 12-cycles of PCR: 98 ̊C for 180 s, 12x (98 ̊C for 15 s, 67 ̊ C for 20 s, 72 ̊C for 60 s), and 72 ̊C for 60 s. The 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit, purified with SPRI beads and resulting DNA library was ligated to the sequencing adapter followed by indexing PCR: 98 ̊C for 45 s; 12x 98 ̊C for 20 s, 54 ̊C for 30 s, 72 ̊C for 20 s), and 72 ̊C for 60 s. The final DNA library was double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina Nova-Seq platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher) at depth of 200-270 million reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Chromium - Single Cell 3’ Reagent Kit (v2)
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| Data processing |
FASTQ files were processed using the 10x Cell Ranger package (v7.01).
Assembly: GRCm38.p5 (mm10)
Supplementary files format and content: CD45neg_thymic_stroma_all+annotation.h5ad: H5AD file with raw and normalized read counts and cell annotation (after bad quality cell filtering)
Supplementary files format and content: filtered_feature_bc_matrix files (barcodes, features, matrix): contain only detected cell-associated barcodes in MEX format; each element of the matrix is the number of UMIs associated with a feature (row) and a barcode (column)
Supplementary files format and content: filtered_feature_bc_matrix.h5: same information as filtered_feature_bc_matrix in HDF5 format
Supplementary files format and content: raw_feature_bc_matrix files (barcodes, features, matrix): contain all detected barcodes in MEX format; each element of the matrix is the number of UMIs associated with a feature (row) and a barcode (column)
Supplementary files format and content: raw_feature_bc_matrix.h5: same information as raw_feature_bc_matrix in HDF5 format
Supplementary files format and content: metrics_summary.csv: metrics for quality assessment in CSV format
Supplementary files format and content: molecule_info.h5: contains per-molecule information for all molecules that contain a valid barcode, valid UMI, and were assigned with high confidence to a gene or Feature Barcode; this file is a required input to run cellranger aggr
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| Submission date |
Aug 03, 2023 |
| Last update date |
Jun 27, 2024 |
| Contact name |
Anastasia I Kousa |
| E-mail(s) |
akousa@coh.org
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| Organization name |
City of Hope
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| Department |
Hematology / HCT
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| Lab |
The Marcel van den Brink Lab
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| Street address |
1500 E Duarte Rd
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| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE240016 |
Age-related epithelial defects limit thymic function and regeneration [single cell] |
| GSE240020 |
Age-related epithelial defects limit thymic function and regeneration |
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| Relations |
| BioSample |
SAMN36828552 |
| SRA |
SRX21240157 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7679887_LJ129_Baseline_EC_filtered_barcodes.tsv.gz |
4.7 Kb |
(ftp)(http) |
TSV |
| GSM7679887_LJ129_Baseline_EC_filtered_feature_bc_matrix.h5 |
1.8 Mb |
(ftp)(http) |
H5 |
| GSM7679887_LJ129_Baseline_EC_filtered_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM7679887_LJ129_Baseline_EC_filtered_matrix.mtx.gz |
3.9 Mb |
(ftp)(http) |
MTX |
| GSM7679887_LJ129_Baseline_EC_metrics_summary.csv.gz |
374 b |
(ftp)(http) |
CSV |
| GSM7679887_LJ129_Baseline_EC_molecule_info.h5 |
15.7 Mb |
(ftp)(http) |
H5 |
| GSM7679887_LJ129_Baseline_EC_raw_barcodes.tsv.gz |
703.6 Kb |
(ftp)(http) |
TSV |
| GSM7679887_LJ129_Baseline_EC_raw_feature_bc_matrix.h5 |
4.7 Mb |
(ftp)(http) |
H5 |
| GSM7679887_LJ129_Baseline_EC_raw_features.tsv.gz |
254.1 Kb |
(ftp)(http) |
TSV |
| GSM7679887_LJ129_Baseline_EC_raw_matrix.mtx.gz |
8.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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