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| Status |
Public on Sep 02, 2022 |
| Title |
SC335 2019-118-LT |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Lung
age: 42
Sex: Female
disease status: SSc
patientcorecode: 2019-118-LT
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Explanted lungs were finely diced and digested for 1 hour in a mix of Liberase DL/DNAse I. A single cell suspension was generated by filtering lungs through 70 micron filters and resuspending cells in PBS containing 0.04% BSA. For some lungs, certain cell populations were partially depleted with Miltenyi magnetic bead separation. For scATAC, cells were lysed according to 10X Genomics' Nuclei Isolation for Single Cell ATAC Sequencing. Cells or nuclei were loaded into the 10X Genomics Chromium instrument per manufacturer's directions.
Libraries were constructed per the manufacturer’s instructions (Single Cell 5' v1, Single Cell 3' v3, scATACv1 protocols, 10x Genomics). In short, for the scRNA protocols, lung cells were added to the master mix and loaded into the chip; subsequently, gel beads and partitioning oil were added into the chip which was then loaded into the Chromium instrument, generating the gel bead-in-emulsion (GEM). Once emulsified, cells were lysed and poly-A RNA transcribed to cDNA, adding an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and 10x Barcode. Silane DynaBeads were then used to clean up the pooled and barcoded cDNA which was then amplified by PCR. SPRIselect reagent was used to select the appropriately sized fragments for library construction. Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added during the library construction protocol. For scATAC, nuclei were incubated for one hour with the transposase then loaded into the Chromium instrument with barcoding reagents, gel beads, and partitioning oil. Once emulsified, the P5 sequence and 10X barcode were added during the PCR GEM generation and barcoding steps. The resulting 10x barcoded single-stranded DNA was then cleaned up with Silane Dynabeads and size selected with SPRIselect reagent. Finally, a P7 sequence and a sample index were added during library construction via PCR.
scRNA and scATAC
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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 |
SC335
10X Genomics 5PrimeV1
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| Data processing |
For scRNA, FASTQ generation, demultiplexing, barcode assignment, gene counting and antibody counting were performed using 10X Genomics' Cell Ranger software v6 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger). For scATAC, FASTQ generation, demultiplexing, barcode assignment, and peak calling were performed using 10X Genomics' Cell Ranger-ATAC software v1.2.0 (https://support.10xgenomics.com/single-cell-atac/software/pipelines/latest/what-is-cell-ranger-atac)
Data were aggregated and analyzed using the R packages Seurat v 4.0 and Signac v 1.0.9004 as well as Loupe Browser 3.1.1 (10X Genomics)
Assembly: GRCh38 and hg19
Supplementary files format and content: 10x Genomics output scRNA files: raw_feature_bc_matrix.h5
Supplementary files format and content: 10x Genomics output scATAC files: fragments.tsv.gz, fragments.tsv.gz.tbi, singlecell.csv, summary.csv, raw_peak_bc_matrix.h5, filtered_peak_bc_matrix.h5, filtered_tf_bc_matrix.h5, peaks.bed, peak_annotation.csv, posssorted_bam.bam.bai
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| Submission date |
Aug 26, 2022 |
| Last update date |
Sep 04, 2022 |
| Contact name |
Tracy Tabib |
| Organization name |
University of Pittsburgh
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| Department |
Department of Medicine
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| Lab |
Laboratory of Robert Lafyatis
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| Street address |
200 Lothrop Street
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15213 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE212109 |
Epigenetic regulation of profibrotic macrophages in systemic sclerosis- associated interstitial lung disease |
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| Relations |
| BioSample |
SAMN30522788 |
| SRA |
SRX17243310 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6509497_SC335raw_feature_bc_matrix.h5 |
20.4 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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