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| Status |
Public on Oct 25, 2019 |
| Title |
Tbx21CreRFPpos |
| Sample type |
SRA |
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| Source name |
spleen
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: spleen
purification: Lin-CD64-Ly6C-CD11C+MHCII+Tbx21CreRFP+
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Spleens were digested in collagenase in RPMI1640 supplemented with 5% fetal calf serum, 1% L-glutamine, 1% penicillin–streptomycin, 10 mM HEPES, 1 mg/ml collagenase A (Sigma, 11088793001) and 1U/mL DNase I (Sigma, 10104159001) for 45 min at 37°C, 250 rpm. Samples were washed and incubated in digest solution for 30 min. Digested samples were filtered through 100-μm strainers and centrifuged to remove collagenase solution. Mouse DC subsets were enriched from a pool of two spleens with Dynabeads™ Mouse DC Enrichment Kit (Invitrogen). Lin(CD3, CD19, CD90)–CD64–Ly6C–CD11c+MHCII+ cells were then purified using an Aria II cell sorter (BD Bioscience) into two populations (RFP+ and RFP– cells). Cells were sorted into cRPMI, before being pelleted and resuspended in RPMI-2% FBS for single-cell RNA seq.
The scRNA-seq libraries were prepared following the user guide manual (CG00052 Rev E) provided by the 10X Genomics and Chromium™ Single Cell 3' Reagent Kit (v2). Briefly, samples were encapsulated in microfluidic droplets at a dilution of ~70 cells/µl (doublet rate ~3.9%.). Encapsulated cells were subjected to reverse transcription (RT) reaction at 53ºC for 60 min. After RT step, the emulsion droplets were broken and barcoded-cDNA was purified with DynaBeads, followed by 14-cycles of PCR-amplification (98 °C for 180 s; [98 °C for 15 s, 67 °C for 20 s, 72 °C for 60 s] x 12-cycles; 72 °C for 60 s). 50 ng of PCR-amplified barcoded-cDNA was fragmented with the reagents provided in the kit and purified with SPRI beads to obtain an average fragment size of 600 bp. Next, the DNA library was ligated to the sequencing adapter followed by indexing PCR (98 °C for 45 s; [98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s] x 10 cycles; 72 °C for 60 s). The resulting DNA library was double-size purified (0.6-0.8X) with SPRI beads and sequenced on an Illumina NovaSeq platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 96 cycles) resulting in 184.5-186.1 million reads per sample (average reads per single-cell being 42,000 and average reads per transcript 4.40-7.14).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
mouse_spleen_counts_normalized_4464x11755.tsv
mouse_spleen_cell_metadata_4464x9.tsv
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| Data processing |
Single-cell RNA seq samples were individually processed using the SEQC v0.2.1 pipeline (Azizi et al., 2018) using the package provided mm38 mouse genome reference or hg38 human genome reference and default parameters for the 10X platform. The SEQC pipeline performs read alignment, multi-mapping read resolution, as well as cell barcode and UMI correction to generate a (cells x genes) count matrix. The pipeline filters empty droplets from true cells based on total molecule counts, filters cells with >20% mitochondrial molecule fraction and filters cells with low library complexity. For each sample, an additional library size filter was used to remove any remaining low quality cell libraries. Genes that were expressed in more than 10 cells were retained for further analysis.
The filtered count matrix was normalized for library size. This normalized matrix was then multiplied by the median of the total molecule count across all cells for numerical stability and log2 transformed with a pseudocount of 0.1 for downstream analysis.
Phenograph was used to cluster PCA reduced expression data for cell type identification. Putative cell doublets were identified using DoubletDetection (gitub.com/JonathanShor/DoubletDetection) and removed from further analysis.
Genome_build: mm38 or hg38
Supplementary_files_format_and_content: tab-delimited text files containing raw counts generated by the SEQC pipeline for each sample, library-size normalized log2-transformed counts for each experiment and associated cell metadata containing per-cell annotations as presented in the publication. The file cell_barcodes.tsv.gz (available on the series record) includes the cell barcode for each cell ID.
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| Submission date |
Sep 19, 2019 |
| Last update date |
Oct 25, 2019 |
| Contact name |
Herman Gudjonson |
| Organization name |
Memorial Sloan Kettering Cancer Center
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| Department |
Computational and Systems Biology Program, SKI
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| Lab |
Dana Pe'er
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| Street address |
417 E 68th St
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE137710 |
Transcriptional basis of mouse and human dendritic cell heterogeneity revealed by single-cell profiling |
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| Relations |
| BioSample |
SAMN12787367 |
| SRA |
SRX6872897 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4085510_mouse_spleen_Tbx21CreRFPpos_raw_counts.tsv.gz |
5.6 Mb |
(ftp)(http) |
TSV |
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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