|
Status |
Public on Nov 02, 2018 |
Title |
HG1-P |
Sample type |
SRA |
|
|
Source name |
serous epithelial ovarian cancer
|
Organism |
Homo sapiens |
Characteristics |
sample id: HG1-P diagnosis: HGSOC site: Primary subject id: 349
|
Growth protocol |
Pathological discard tissue, from women who were scheduled for surgery to evaluate a suspicious pelvic mass, primary tumor and metastatic lesions (when available), were obtained at time of surgery and frozen for later analysis. Tissue was minced and frozen in 40% FBS, 40% RPMI and 20% DMSO.
|
Extracted molecule |
polyA RNA |
Extraction protocol |
Thawed tissue samples were digested and dissociated into single-cell suspensions using Miltenyi’s gentleMACS Octo dissociator and tumor dissociation kit following manufacturer’s instructions. Single cells were collected by straining digested tissue through a MACS Smart strainer (70um), washed, then layered over a Ficoll gradient to remove red blood cells and debris. To enrich the tumor cell fraction for live cells, dead cells were excluded using Dead Cell Removal kit and MS columns (Miltenyi Biotec) and remaining viable cells were prepared for scRNA-seq. Using the BioRad droplet digital SEQ Single-cell Isolator and the Illumina SureCell Whole Transcriptome Analysis 3’ library prep kit, scRNA-seq was performed on isolated cells from ovarian tumor tissue samples following the manufacturer’s instructions. Briefly single-cells were encapsulated, lysed then barcoded within each droplet. Following first and second strand cDNA synthesis, Illumina’s Nextera technology was used to generate a library for NGS. Final libraries were assessed and quantified using a High Sensitivity DNA chip and a 2100 BioAnalyzer (Agilent Technologies) prior to sequencing on a NexSeq 500 high output flow cell.
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Data processing |
Sample data was demultiplexed and raw sequence files generated using bcl2fastq2 app in Illumina’s cloud application, Basespace. SureCell RNA Single-Cell application to extract the cell barcodes and the Unique Molecular Identifiers (UMIs). The raw sequence files were aligned to the hg19 human reference genome and gene expression was quantified. A knee plot of the genic UMIs per cell barcode was used to set thresholds for cells for downstream analysis. Genome_build: hg19 Supplementary_files_format_and_content: umicount table with genes as columns and cell barcodes as rows
|
|
|
Submission date |
Aug 21, 2018 |
Last update date |
Nov 03, 2018 |
Contact name |
Andrew Shih |
E-mail(s) |
ashih@northwell.edu
|
Phone |
516-562-2960
|
Organization name |
Feinstein Institute - Northwell Health
|
Street address |
350 Community Dirive
|
City |
Mahasset |
State/province |
NY |
ZIP/Postal code |
11030 |
Country |
USA |
|
|
Platform ID |
GPL18573 |
Series (1) |
GSE118828 |
Identification of grade and origin specific cell populations in serous epithelial ovarian cancer by single cell RNA-seq |
|
Relations |
BioSample |
SAMN09869249 |
SRA |
SRX4582172 |