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| Status |
Public on Apr 26, 2021 |
| Title |
CRISPR_Donor3_IL2_high |
| Sample type |
SRA |
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| Source name |
CD4+CD25- T cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD4+CD25- T cells
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| Treatment protocol |
CD4+CD25- T cells were infected with lentivirus containing a 6000 guide RNA CRISPR library. Cas9 ribonucleoprotein complex was electroporated. 6 days after electroporation, FACS was performed to isolate CD25, IL-2, or CTLA4 high and low expressing cells.
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| Growth protocol |
Cells were grown in RPMI (Sigma, Cat # R0883) with 10% FCS (Sigma, Cat # F0926), with 100U/mL Pen-Strep (Gibco, Cat # 15140-122), 2mM L-Glutamine (Sigma, Cat # G7513), 10mM HEPES (Sigma, Cat # H0887), 1X MEM Non-essential Amino Acids (Gibco, Cat # 11140-050), 1mM Sodium Pyruvate (Gibco, Cat # 11360-070), and 50 U/mL IL-2 (Amerisource Bergen, Cat #10101641) at a concentration of 1E6 cells/mL.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After sorting, cells were washed with PBS, counted, pelleted, and resuspending at up to 5E6 cells per 400 µl of lysis buffer (1% SDS, 50 mM Tris, pH 8, 10 mM EDTA). The remaining protocol reflects additives/procedures performed per each 400 µl of sample. 16µl of NaCl (5M) was added, and the sample was incubated on a heat block overnight at 66°C. The next morning, 8µl of RNAse A (10mg/ml, resuspended in ddH2O) (Zymo, Cat #E1008) was added, and the sample was vortexed briefly, and incubated at 37°C for 1 hour. Next, 8µl of Proteinase K (20mg/ml) (Zymo, Cat #D3001) was added, the sample was vortexed briefly, and incubated at 55°C for 1 hour. A phase lock tube (Quantabio, Cat #2302820) was prepared for each sample by spinning down the gel to the bottom of the tube at 20,000g for 1 minute and then 400µl of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) was added to each tube. 400µl of the sample was then added to the phase lock tube and the tube was shaken vigorously. The sample was centrifuged at maximum speed at room temperature for 5 minutes. The aqueous phase was transferred to a low-binding eppendorf tube (Eppendorf, Cat #022431021) and then 40µl of Sodium Acetate (3M), 1µl GlycoBlue (Invitrogen, Cat # AM9515), and 600µl of room temperature isopropanol was added. The sample was then vortexed and stored at -80°C for 30 minutes or until the sample had frozen solid. Next the sample was centrifuged at maximum speed at 4°C for 30 minutes, the pellet was washed with fresh 70% room temperature Ethanol, and allowed to air dry for 15 minutes. Pellets were then resuspended in Zymo DNA elution buffer (Zymo, Cat No: D3004-4-10), and placed on the heat block at 65°C for 1 hour to completely dissolve the genomic DNA.
sgRNA was amplified and barcoded from the genomic DNA according to the protocol by Joung et al. (Joung et al. 2017). Up to 2.5 µg of genomic DNA were added to each 50 µL reaction, which included 25 µL of NEBNext Ultra II Q5 master mix (NEB, Cat #M0544L), 1.25 µL of the 10 µM forward primer and 1.25 µL of the 10 µM reverse primer, and H2O to 50 uL. The following PCR cycling conditions were used: 98°C for 3 minutes, followed by 23 cycles at 98°C for 10 seconds, 63°C for 10 seconds, and 72°C for 25 seconds, and ending with 2 minutes at 72°C. Samples were then cleaned and concentrated in Zymo Spin-V columns (Zymo, Cat #C1016-50) following Joung et al., and eluted in 150 uL of Zymo DNA elution buffer. Up to 2 µg of each library were loaded on a 2% agarose gel, and the band at ~250 base pairs was extracted using the Zymoclean Gel DNA recovery kit (Zymo, Cat #D4008). The concentration of each sample was then measured using the Qubit dsDNA high sensitivity assay kit (Thermo Fisher Scientific, Cat #Q32854). Samples were then sequenced on an Illumina HiSeq 4000 using 10-30% PhiX (Illumina, Cat #15017872), and a custom sequencing primer.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Library strategy: CRISPR sequencing
A table of individual guide abundance in each sample was generated using the count command in MAGeCK version 0.5.8
Genome_build: None
Supplementary_files_format_and_content: Count of guide RNAs.
Supplementary_files_format_and_content: raw counts
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| Submission date |
Apr 08, 2021 |
| Last update date |
Apr 26, 2021 |
| Contact name |
Alexander Marson |
| E-mail(s) |
[email protected]
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| Organization name |
Gladstone-UCSF Institute of Genomic Immunology
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| Street address |
1650 Owens St
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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) |
| GSE171674 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [CRISPR] |
| GSE171737 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks |
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| Relations |
| BioSample |
SAMN18672097 |
| SRA |
SRX10549772 |
| 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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