|
Status |
Public on Jun 21, 2018 |
Title |
HEK293T_rs199643834_EDIT_HET [2_70] |
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.
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
|
|
Description |
2_70
|
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.
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|
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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 |
SAMN09461949 |
SRA |
SRX4277126 |