|
| Status |
Public on Jul 02, 2020 |
| Title |
HEK293T cells, nCas9_RNF2_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
treatment: nCas9
guide rna (grna): RNF2
gfp: All GFP
replicate: 2
|
| Treatment protocol |
Transfections were performed using 50ug of transfection quality plasmid DNA (Qiagen Maxiprep), using 37.5ug of base editor plasmid co-translationally encoding P2A-EGFP or variants thereof and 12.5ug of gRNA plasmids. 6-7x10E6 HEK293T cells were seeded into TC-treated 150mm plates 18-24h prior to transfection to yield ~60-80 % confluency on the day of transfection. Cells were transfected using 150uL TransIT-293 (HEK293T, Mirus) reagents per plate, according to the manufacturers’ protocols (mixing DNA and reagent in 5mL Optimem, 30min incubation). GFP positive cells were sorted 36-40h after transfection after doublet-exlusion and gating for the cell population on a BD FACSARIA II.
|
| Growth protocol |
HEK293T cells (CRL-3216, obtained from ATCC) were grown in culture using media consisting of DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Gibco). Cells were used for experiments until passage 20. Cells were passaged at ~80% confluency every 2-4 days to maintain an actively growing population and avoid anoxic conditions. Cells were tested for mycoplasma bi-weekly.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Macherey-Nagel’s NucleoSpin RNA Plus kit.
Illumina TruSeq Stranded Total RNA Gold, IDT-ILM unique dual indices for barcoding.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
RNA sequencing data was aligned to the human reference genome (GRCh38/hg38) using STAR using a basic two-pass alignment strategy. Aligned reads were filtered for those reads that were uniquely mapping using the --outFilterMultimapNmax 1 flag.
Variants associated with BE treatment were called using the GATK best practices pipeline using GATK 3.8.1 and Picard 2.7.
No samples were excluded from bioinformatics analyses.
Genome_build: hg38
Supplementary_files_format_and_content: Raw gene expression counts per gene estimated by STAR
|
| |
|
| Submission date |
Sep 13, 2019 |
| Last update date |
Jul 02, 2020 |
| Contact name |
Caleb Lareau |
| E-mail(s) |
[email protected]
|
| Organization name |
Memorial Sloan Kettering
|
| Street address |
417 E 68th St, Zuckerman - ZRC 1132
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE137411 |
Bimodal Adenine and Cytosine CRISPR Base Editing on DNA |
|
| Relations |
| BioSample |
SAMN12744784 |
| SRA |
SRX6845789 |
