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| Status |
Public on Dec 16, 2020 |
| Title |
a549_day0_rep1 |
| Sample type |
SRA |
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| Source name |
a549_day0
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| Organism |
Homo sapiens |
| Characteristics |
cell line background: A549
source tissue: Lung
genotype/variation: dCas9-KRAB expressing
time point: day0
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| Treatment protocol |
To introduce expressiond of dCas-KRAB, each cell line was titrated with library virus generated from hU6-sgRNA hUbC-dCas9-KRAB-T2a-Pur (Addgene plasmid # 71236). MOI was determined by infecting ~5-6 million cells with varying amounts of library virus for 24 hours, which were then split into media with or without puromycin for 48-72 hours (A549, 3.5 ug/mL; 22Rv1, V16A, 3 ug/mL; LNCaP, 2.5 ug/mL). A ratio between these two populations was calculated to determine the infection efficiency to achieve a MOI of ~0.3. The amount of library virus was scaled up along with the number of cells to ensure that on average every sgRNA was represented in ~300 cells. For each screen, cells were split into triplicates every 3-4 days, and maintained at 300x coverage throughout the screen.
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| Growth protocol |
A549, 22Rv1 and V16A cells were cultured in RPMI1640 medium with 10% FBS (Wisent) and 1% Penicillin and Streptomycin (450-201-EL, Wisent). All cells were cultured at 37 degrees in 5% CO2. All cell lines were authenticated by STR and routinely tested for mycoplasma using the EZ-PCR mycoplasma Test Kit.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were collected in replicates on Day 0 and Day 16 post- puromycin selection for genomic DNA analysis.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
guide RNA
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| Data processing |
The fastq files were first converted to fasta file using custom shell script. For each sample, a custom bowtie database was generated by the command bowtie-build in bowtie suite (version 1.1.2). The library sgRNAs were mapped against the database for each sample using bowtie with the parameter v=0 and default values for other parameters.
Each targeted region in the library was split into 100 bp sliding windows (50 bp offset) ensuring at least two sgRNAs target a window. The differential sgRNA abundance was estimated using the tool MAGeCK (Li et al., 2014). The 100 bp window with the lowest p-value in each CRE is treated as a representative of the essentiality of the CRE. For adjacent 400 bp windows, we merge all windows into the most essential window. The depletion score of each CRE is the “neg.score” as reported by MAGeCK.
Genome_build: Hg19
Supplementary_files_format_and_content: MAGeCK output for all cell lines. The first column is the library region, the next nine columns indicate the p value, log2 fold change and depletion score of differential analyses between day 16 and 0 in V16A, 22Rv1 and A549 cells, respectively. The last column indicate the class of the region (CRE or control regions).
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| Submission date |
Jan 01, 2020 |
| Last update date |
Dec 16, 2020 |
| Contact name |
Musaddeque Ahmed |
| E-mail(s) |
musaddeque.ahmed@gmail.com
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| Organization name |
University health Network
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| Lab |
He Lab
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| Street address |
101 College St
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| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5G1L7 |
| Country |
Canada |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE142808 |
CRISPRi screen of prostate cancer associated risk cis-regulatory elements [CRISPRi] |
| GSE142811 |
CRISPRi screen of prostate cancer associated risk cis-regulatory elements |
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| Relations |
| BioSample |
SAMN13705328 |
| SRA |
SRX7484897 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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