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SRX17243305: GSM6509492: SC313 2019-095-CORE; Homo sapiens; RNA-Seq
1 ILLUMINA (Illumina NovaSeq 6000) run: 130.6M spots, 11.9G bases, 2.8Gb downloads

External Id: GSM6509492_r1
Submitted by: Laboratory of Robert Lafyatis, Department of Medicine, University of Pittsburgh
Study: Epigenetic regulation of profibrotic macrophages in systemic sclerosis- associated interstitial lung disease
show Abstracthide Abstract
Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is the leading cause of death in patients with systemic sclerosis (SSc) with unclear pathogenesis and limited treatment options. Evidence strongly supports an important role for profibrotic, SPP1-expressing macrophages in SSc-ILD. We sought to define the transcriptome and chromatin structural changes of SPP1 SSc-ILD macrophages, so as to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype. Overall design: We performed single cell RNA-sequencing on 11 explanted SSc-ILD and healthy control lung samples, as well as single cell ATAC-sequencing on a subset of lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using SCENIC and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat.
Sample: SC313 2019-095-CORE
SAMN30522793 • SRS14808432 • All experiments • All runs
Organism: Homo sapiens
Library:
Name: GSM6509492
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC SINGLE CELL
Selection: cDNA
Layout: PAIRED
Construction 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
Runs: 1 run, 130.6M spots, 11.9G bases, 2.8Gb
Run# of Spots# of BasesSizePublished
SRR21233225130,572,72111.9G2.8Gb2022-09-02

ID:
24063299

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