|
| Status |
Public on Dec 19, 2020 |
| Title |
EpCAM-positive |
| Sample type |
SRA |
| |
|
| Source name |
PC9 U937 mix
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC9 U937 mix
treatment: 4.725x106 mixed cells were separated with EpCAM beads. EpCAM+ fraction containing epithelial cells was collected and used for Drop-seq
timepoint: Cells selected using CD326 (EpCAM) MicroBeads (Miltenyi Biotec, cat#130-061-101).
|
| Treatment protocol |
1.5x106 PC9 cells was seeded on a p150 plate. 2.5x105 U937 cells were seeding per ml in 30 mL of media in a 162 cm2 flask, and 3 uL of 1 mM of TPA was added to induce differentiation. Cells were trypsinized, centrifuged and resuspended in RPMI with FBS, counted, and processed further for separation with Milteny microbeads and Drop-seq.
|
| Growth protocol |
Non-small cell lung carcinoma cell line PC9 (Sigma) was grown in RPMI, 5% FBS (HyClone) media containing penicillin and streptomycin (1% PS) in 5% CO2 at 37ºC. Cells obtained from vendor were maintained for 8 passages before collection for scRNA analysis. U937 cells were grown in RPMI, 10% FBS (HyClone), 1% PS, in 5% CO2 at 37ºC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Drop-seq on the single cell suspension
Nextera XT tagmentation using Illumina cat# FC-131-1096 (Drop-seq protocol)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Single cell RNA-seq was performed using Drop-seq
|
| Data processing |
Illumina Casava1.7 software used for basecalling.
Illumina paired end raw FastQ file were processed for read alignment and gene expression. Drop-seq single-cell data was analyzed using the data anlysis protocol described in Drop-seq cook-book (Macosko et al. 2015)(http://mccarrolllab.com/dropseq/) and used the Drop-seq_tools-1.13. We used STAR aligner to align the reads against genome version GRCh37.p13 hg19 (From Ensembl) and corresponding gene model is extracted from Ensembl version 74.
Quality of read and mapping were checked using the program FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
Digital Gene Expression (DGE) data obtained from an aligned library is done using the Dropseq program DigitalExpression (integrated in Drop-seq_tools-1.13). Number of cells that were extracted from aligned BAM file is based on knee plot which extracts the number of reads per cell, then plot the cumulative distribution of reads and select the “knee” of the distribution.
Genome_build: GRCh37.p13
Supplementary_files_format_and_content: Tab-delimited text file. Gene expression (as count) of each gene for each extracted cell
|
| |
|
| Submission date |
Jul 25, 2019 |
| Last update date |
Dec 19, 2020 |
| Contact name |
Elizaveta V Benevolenskaya |
| E-mail(s) |
[email protected]
|
| Phone |
312-413-8947
|
| Organization name |
University of Illinois at Chicago
|
| Department |
Biochemistry and Molecular Genetics
|
| Street address |
900 S. Ashland Ave.
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60607 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE134842 |
Assessing efficiency of single cell population by Drop-seq using cell mixture of epithelial and myeloid cells. |
| GSE149383 |
Drug resistant changes at the level of single cells |
|
| Relations |
| BioSample |
SAMN12360854 |
| SRA |
SRX6589350 |
