|
| Status |
Public on Nov 19, 2022 |
| Title |
Sorted-Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
HCT116 cells with integrated reporter
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
cell type: colorectal cancer cell
library: Brunello lentiviral library
gfp expression: sorted cells (those with high GFP signal)
biological replicate: 2
|
| Treatment protocol |
The cells were transduced with the Brunello lentiviral library (100X) and two days later puromycin was added. Seven days postinfection the hightes 8% GFP expressing cells were selected using a FACSAria cell sorter. Unselected cells were taken as a reference.
|
| Growth protocol |
An HCT116-derived clonal line that expresses the ORF59-GFP reporter was grown in DMEM supplemented with 10% fetal bovine serum and 1X penicillin-streptomycin (Sigma), and 2mM L-glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted using phenol:chloroform:iso-amyl alcohol method. See Scarborough et al 2021 for details.
Library amplification was completed following a two-step variation of the BROAD institute protocol (BROAD Institute PCR of sgRNAs for Illumina sequencing). The two step variation of the BROAD institute protocol consists of an initial amplification step using primers flanking the P5 and P7 primers used for sequencing.
CRISPR screen
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
DNA-lentiviral sgRNA sequencing
These are sgRNAs sequenced from selected high-GFP expressing cells
AMD_3rep.gene_summary.txt
AMD_3rep.sgrna_summary.txt
|
| Data processing |
The fastq files were subjected to quality check using fastqc (version 0.11.9, http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and adaptors trimmed using Cutadapt version 3.3 (Martin M. et al. 2011)
The trimmed fastq files were mapped to Brunello library sequence (Doench et al. 2016; Addgene #73179), read counts generated, and median normalized using MAGeCK-VISPR version 0.5.6 (Li W. et al. 2015) disallowing any mismatches
Batch correction was performed in MAGeCK-VISPR after excluding zero count sgRNAs, considering each biological replicate as a separate batch
Positively and negatively selected sgRNA and genes were identified using the default parameters of MAGeCK-VISPR.
Assembly: n/a; seqences aligned to Brunello (Doench et al. 2016; Addgene #73179)
Supplementary files format and content: text file; output for MAGeCK
|
| |
|
| Submission date |
Apr 18, 2022 |
| Last update date |
Nov 19, 2022 |
| Contact name |
Nicholas K Conrad |
| E-mail(s) |
nicholas.conrad@utsouthwestern.edu
|
| Phone |
2146480795
|
| Organization name |
UT Southwestern Medical Center
|
| Department |
Microbiology
|
| Street address |
6000 Harry Hines Blvd
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75063 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE200991 |
The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication [CRISPR] |
| GSE201046 |
The PNUTS-protein phosphatase 1 complex acts as an intrinsic barrier to KSHV replication |
|
| Relations |
| BioSample |
SAMN27622607 |
| SRA |
SRX14894045 |
