|
| Status |
Public on Nov 15, 2024 |
| Title |
GIN435.WNK2/WNK3-T15B |
| Sample type |
SRA |
| |
|
| Source name |
Blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell line: HAP1
cell type: chronic myelogenous leukemia
genotype: WNK2_WNK3 dual mutant
treatment: Genome-wide CRISPR screen with TKOv3
|
| Treatment protocol |
The Toronto knockout (TKOv3) genome-scale CRISPR library (Addgene #90294) was used to perform pooled CRISPR knockout screens in HAP1 parental and knockout cell lines to identify genetic interactions. For each screen, a total of 9E7 cells were transduced with TKOv3 lentivirus at an MOI of 0.3 and > 200-fold library coverage. 24 hours after infection, transduced cells were selected with a medium containing 1 μg/ml puromycin for 48 hours. Cells were then harvested, pooled, and split into three replicates of at least 1.5 x 107 cells maintaining 200-fold library coverage. Approximately 3 – 5 x 107 cells were also collected for gRNA extraction and determination of library representation at day 0 (T0 reference). Cells were passaged every 3-4 days at > 200-fold library coverage until 15 population doublings were reached. Cell pellets for gRNA extraction were collected at every cell passage for each replicate. For WNK463 drug-treated screens, replicates were separated into drug or vehicle (DMSO, <0.1% v/v) treatment screening arms at passage T4. Both WNK463-treated and DMSO control replicates were passaged at > 200-fold coverage on the same day until drug dosing at IC50 reached three rounds. Cell pellets for treated and control replicates were also collected at each passage.
|
| Growth protocol |
HAP1 cells (Horizon Discovery, male human) were maintained in IMDM (Wisent) supplemented with 10% FBS (Gibco) and as needed supplemented with 1% penicillin–streptomycin (PS) (Gibco). HAP1 cells were maintained at 37°C with 5% CO2 and passaged regularly and were dissociated with 0.25% trypsin-EDTA (Gibco) for passaging. HAP1 cells were regularly monitored for mycoplasma contamination using the e-Myco PLUS Mycoplasma PCR Detection kit (iNtRON Bio).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
gRNA abundance was measured at T0 and endpoints of each screen by next-generation sequencing (NGS). Genomic DNA was extracted from cell pellets using the Wizard Genomic DNA Purification kit (Promega).
Sequencing libraries were prepared from 50 µg gDNA (200-fold library coverage of haploid genome) via a 2-step nested PCR using primers that include Illumina TruSeq adapters with i5 and i7 indices. Barcoded libraries were gel purified using PureLink Quick Gel Extraction kit (Thermo Fisher Scientific) and sequenced on an Illumina HiSeq2500 or NovaSeq6000 using single-read sequencing and standard primers for dual-indexing with HiSeq SBS Kit v4 or NovaSeq v1.5 reagents. T0 samples were sequenced at read depths of 500-fold library coverage, while all other samples were sequenced at 200-fold. Amplicons were sequenced using a custom sequencing recipe comprising DC: 21 cycles; R1: 36 cycles; Index read 1: 8 cycles; Index read 2: 8 cycles.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
sgRNA amplified from gDNA
GIN_6460
|
| Data processing |
Reads from each sample were mapped to the TKOv3 reference library using Bowtie (v0.12.8) allowing up to two mismatches and one exact alignment (specific parameters: -v2, -m1, -p4 -sam-nohead).
Successfully aligned reads were counted and combined into a matrix with guide annotations.
Read counts were normalized to 10 million reads per sample and log fold-changes (LFC) of each gRNA between each timepoint and T0 were determined. Guides with fewer than 30 or greater than 10,000 absolute read counts at T0 were removed.
To identify genetic interactions, the qGI algorithm was used to score the differential effects between HAP1 parental (Arreger et. al.) and gene knockout screens, while the Bioconductor package limma was used to examine drug-treated versus DMSO effects. Interactor genes with FDR (or minimum FDR across replication screens) > 0.2 were plotted with GraphPad (PRISM software).
Assembly: hg38
Supplementary files format and content: tab-delimited read count matrix
Library strategy: CRISPR screen
|
| |
|
| Submission date |
Jun 10, 2024 |
| Last update date |
Nov 15, 2024 |
| Contact name |
Jason Moffat |
| E-mail(s) |
[email protected]
|
| Organization name |
Hospital for Sick Children
|
| Department |
Genetics and Genome Biology
|
| Lab |
Moffat
|
| Street address |
686 Bay Street
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G0A4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE269526 |
TSC22D, WNK and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation |
|
| Relations |
| BioSample |
SAMN41779442 |
| SRA |
SRX24865308 |
