|
| Status |
Public on Jan 25, 2022 |
| Title |
sgRNA-enrichment_20k-sgRNAs_Lane6_NovaSeq |
| Sample type |
SRA |
| |
|
| Source name |
MDA-MB-231 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231 cells
experiment: Single-cell Expression, sgRNA enrichment
lane: Lane6
|
| Treatment protocol |
A lentivirus library containing 20139 sgRNAs was constructed and packaged by transfecting HEK293T cells. The virus was collected 48 hours after transfection and concentrated by using Lenti-X lentivirus concentrator. For virus infection, 5X10^4 MDA-MB-231 cells were seeded in 24 well plate per well with 500 µl complete medium plus 1µg/ml polybrene. The following day, the medium was changed to fresh complete medium with antibiotics to screen for infected cells.
|
| Growth protocol |
MDA-MB-231 cells were cultured in alpha modified MEM plus 10% FBS, 1mM sodium pyruvate, 10mM HEPES, 1X Glutamax supplement, 1X MEM non-essential amino acid, 1 mg/mL insulin, 1ng/mL hydrocortisone, 25 ug/mL EGF and pen/strep at 37C and 5% CO2.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were collected, washed once with PBS. Then the cells were stained with Cell Hashing antibodies and resuspended in PBS containing 0.05% BSA to the desired concentration and immediately load on the 10X Chromium controller for the single-cell RNA-seq preparation.
For single-cell library preparation, we follow the standard protocol of 10X Chromium 3’ V3 kit. After the cDNA amplification, 50ng of samples were separated for the sgRNA enrichment library preparation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
custom pipeline
Mapping and library combination were performed by using 10X Cellranger V3.1.0
Genome_build: hg38
Supplementary_files_format_and_content: summary of UMI count of each sgRNA for every cell barcode
|
| |
|
| Submission date |
Oct 15, 2021 |
| Last update date |
Jan 25, 2022 |
| Contact name |
Yihan Wang |
| E-mail(s) |
[email protected]
|
| Organization name |
UT Southwestern
|
| Street address |
5323 Harry Hines Blvd.
|
| City |
Dallas |
| State/province |
Texas |
| ZIP/Postal code |
75390 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE185995 |
Computational identification of clonal cells in single-cell CRISPR screens |
|
| Relations |
| BioSample |
SAMN22328998 |
| SRA |
SRX12633411 |
