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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 01, 2024 |
| Title |
wt_2_down |
| Sample type |
SRA |
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| Source name |
Reference, Low expression
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Jurkat
egfp reporter: native RORC switch
gfp level: low
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| Treatment protocol |
The sgRNA library was transduced into Jurkat cells with a multiplicity of infection (MOI) of less than 0.3, resulting in approximately 30% BFP+ cells. After transduction, the cells were transplanted at an average coverage of 500x (Day 0). Selection with 1 µg/mL puromycin was conducted at 48 and 72 hours post-transduction to enrich for positively transduced cells.
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| Growth protocol |
The Jurkat cell line was cultured in RPMI-1640 medium supplemented with 10% FBS, glucose (2 g/L), L-glutamine (2 mM), 25 mM HEPES, penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (1 μg/mL) (Gibco).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
On Day 11, cells were collected and sorted using the BD FACSAria™ Fusion cell sorter. Sorting criteria included the top and bottom 25% of cells based on the ratio of GFP to mCherry fluorescence intensity. The CRISPRi screen was conducted under two conditions using cells with either a reference RORC element - GFP reporter or a mutated 77-AG RORC element - GFP reporter, with each condition tested in duplicate.
After sorting, genomic DNA was harvested (Macherey-Nagel Midi Prep kit) and amplified using NEB Next Ultra II Q5 master mix and primers containing TruSeq Indexes for NGS analysis. Sample libraries were prepared and sequenced on a HiSeq 4000.
The library preparation for CRISPRi/a-v2 involves amplifying sgRNA sequences, which are then sequenced on an Illumina HiSeq 4000 system. The library includes TruSeq indices for demultiplexing post-sequencing. The PCR amplification incorporates Illumina adapters and a unique sample index, facilitating the sequencing of pooled samples in a single sequencing run with a phiX spike-in of 10% to enhance sequence diversity and read accuracy.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
wt_2_down
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| Data processing |
Guides were then quantified with the published ScreenProcessing (https://github.com/mhorlbeck/ScreenProcessing) method and phenotypes generated with an in-house processing pipeline, iAnalyzer (https://github.com/goodarzilab/iAnalyzer). iAnalyzer relies on fitting a generalized linear model to each gene. Coefficients from this GLM were z-score normalized to the negative control guides and finally the largest coefficients were analyzed as potential hits. For the comparison of gene phenotypes between the two cell lines, the DESeq2 ratio of ratios test was used.
Assembly: The analysis does not involve a standard genome build but utilizes a custom reference composed of synthesized oligonucleotide sequences tailored to the experimental design.
Supplementary files format and content: Tab-separated files with raw read counts for each sgRNA.
Library strategy: CRISPR screen
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| Submission date |
Apr 28, 2024 |
| Last update date |
Jul 01, 2024 |
| Contact name |
Hani Goodarzi |
| Organization name |
UCSF
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| Department |
Biochemistry and Biophysics
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| Street address |
600 16th St, GH S312D
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE266057 |
CRISPRi Screen for Investigating the RORC RNA Switch Mechanisms in Jurkat Cells |
| GSE266070 |
RORC RNA Switch Mechanisms |
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| Relations |
| BioSample |
SAMN41105993 |
| SRA |
SRX24387164 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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