|
| Status |
Public on Jun 21, 2018 |
| Title |
HEK293T_rs199643834_EDIT_HET [13_21] |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T_rs199643834_EDIT_HET
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 293T
rs199643834 genotype: ALT/REF/REF
|
| Treatment protocol |
For transfection with Cas9- and sgRNA-expressing plasmids as well as ssODN template, cells were harvested for seeding at a log growth phase (approximately 70% confluency). In a 6-well format, 300,000 293T cells were seeded a day prior to transfection. The next day 2 μg of each lentiCRISPR v2 plasmid and 0.5 μg of ssODN HDR template were delivered into the cells using Lipofectamine 3000 reagent (ThermoFisher Scientific, cat. # L3000008). At 24-hours post-transfection selective pressure in the form of 5 μg/ml puromycin was applied for 8 hours to enrich for transfected cells. This population was selected for single-cell sorting in 96-well format on SONY SH800 to obtain monoclonal edited cell lines which were genotyped.
|
| Growth protocol |
Human 293T cell line (ATCC, cat. # CRL-3216) was adapted to and subsequently routinely grown in Opti-MEM/5% CCS (newborn calf serum), 1% GlutaMAX, 1% Penicillin/Streptomycin and sodium pyruvate. Expanded lines were grown to 70-80% confluency before harvesting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using the Qiagen RNAeasyMini kit.
RNA sequencing libraries were prepared using the TruSeq Stranded mRNA Library Sample Preparation Kit in accordance with manufacturer’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
13_21
|
| Data processing |
Trimmomatic v0.36 was used to clip Illumina adaptors and quality trim.
Reads were aligned to hg19 using STAR in 2 pass mode.
featureCounts v1.5.3 was used in read counting and strand specific mode (-s 2) with primary alignments only to generate gene level read counts with Gencode v19 annotations used in GTEx v6p.
Genome_build: hg19
Supplementary_files_format_and_content: Expression matrix where lines are genes, columns are samples, and the values are the featureCounts read counts.
|
| |
|
| Submission date |
Jun 20, 2018 |
| Last update date |
Jun 22, 2018 |
| Contact name |
Stephane Emile Castel |
| E-mail(s) |
scastel@nygenome.org
|
| Organization name |
New York Genome Center
|
| Lab |
Lappalainen
|
| Street address |
101 Avenue of the Americas
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10013 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE116061 |
Modified penetrance of coding variants by cis-regulatory variation contributes to disease risk |
|
| Relations |
| BioSample |
SAMN09461946 |
| SRA |
SRX4277129 |
