|
| Status |
Public on Jun 24, 2024 |
| Title |
Optimization;RPE1;LbCas12a_2NLS;T21C |
| Sample type |
SRA |
| |
|
| Source name |
cell line
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cell line
cell line: RPE1
cell type: retinal pigment epithelial cells
genotype: TP53 -/-
treatment: lenti virus infected
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from the cell pellets collected at the start (T0) and end time points using the Wizard Genomic DNA Purification Kit (Promega #A1120).
First, the lentiviral integrated hgRNA expression cassettes were amplified from genomic (g)DNA equivalent of ~ 100-fold library coverage using NEBNext Ultra II Q5 Master Mix (New England Biolabs #M0544X). Individual 50 µL PCR reactions containing 3.5 μg gDNA were performed using PCR1_Hybrid_Outer_F2 and PCR1_Hybrid_Outer_R1 primers for the optimization and exon deletion screens and primers A265 and PCR1_BE_Rev for the base editor screens, respectively (see Table S3). After pooling the individual PCR-1 reactions, a fraction was purified using a PCR purification column and used as template for PCR-2, in which each sample was barcoded with unique i5 and i7 index primer combinations. The resulting PCR-2 products were resolved on a 2% agarose gel using SYBR Safe DNA stain (ThermoFisher Scientific #S33102). The desired band was excised and subjected to gel extraction (ThermoFisher Scientific #K0691). Quantification of the extracted libraries was conducted using the Qubit dsDNA HS assay (ThermoFisher Scientific #Q32851) and Tape Station (Agilent #5067-5582 and #5067-5583). The quantified and validated sequencing libraries were pooled, and paired-end sequencing was performed on either an Illumina NextSeq 500 or a NovaSeq 6000 platform.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
optimization_readCounts.txt
|
| Data processing |
demultiplexing
mapping to library with bowtie 1 and extract read count for each gRNA in the library
Assembly: hg18
Supplementary files format and content: read counts of the mapped sequencing data in the format of txt.
Library strategy: CRISPR
|
| |
|
| Submission date |
Sep 29, 2023 |
| Last update date |
Jun 24, 2024 |
| Contact name |
Thomas Gonatopoulos-Pournatzis |
| E-mail(s) |
thomas.gonatopoulos@nih.gov
|
| Organization name |
NCI/NIH
|
| Street address |
1050 Boyles Street
|
| City |
Frederick |
| ZIP/Postal code |
21702 |
| Country |
USA |
| |
|
| Platform ID |
GPL30173 |
| Series (2) |
| GSE244337 |
Genome-Scale Exon Perturbation Screens Uncover Critical Exons for Cell Fitness [CRISPR] |
| GSE244374 |
Genome-Scale Exon Perturbation Screens Uncover Critical Exons for Cell Fitness |
|
| Relations |
| BioSample |
SAMN37607714 |
| SRA |
SRX21932083 |
