 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 13, 2021 |
| Title |
CRISPRi_DolcettoSetA_Donor1_IL2_high |
| Sample type |
SRA |
| |
|
| Source name |
Primary CD4+ T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Primary CD4+ T cells
infection: CRISPRi_DolcettoSetA
sorting: IL2_high
|
| Treatment protocol |
One day after activation, T cells were infected with dCas9-VP64 (CRISPRa) or dCas9-KRAB (CRISPRi) lentivirus. Two days after activation, T cells were split into two populations and infected with Calabrese Set A (addgene 92379) or Calabrese Set B (addgene 92380) lentivirus for CRISPRa, or Dolcetto Set A (addgene 92385) or Dolcetto Set B (addgene 92386) lentivirus for CRISPRi, each at an MOI of 0.5. The following day 2ug/ml puromycin was added to the culture medium and cells were passaged every two days maintaining a minimum concentration of 0.3e6cells/ml. Eight days after initial activation, cells were harvested, spun down and resuspended at 2e6 cells/ml X-VIVO 15 without supplements. Next day, cells were restimulated with anti-CD3/CD28/CD2 immunocult and treated with Brefeldin A for 9 hours, and fixed/stained for FACS. Cells were sorted into IL-2 low and high CD4+ T cells and IFN-γ low and high CD4- T cells
|
| Growth protocol |
Primary human bulk T cells were cultured in X-VIVO 15 (Lonza Bioscience cat 04-418Q) supplemented with 5% FCS, 55mM 2-mercaptoethanol, 4mM N-Acetyl L-Cysteine, and 500 IU/ml recombinant human IL-2 (Amerisource Bergen cat 10101641). Primary T cell activation was done by anti-humanCD3/CD28 CTS dynabeads (Fisher Scientific cat 40203D) at a 1:1 cell to bead ratio at 1e6 cells/ml.
|
| 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 barcodes were amplified from genomic DNA using Takara Ex-Taq polymerase (Takara RR001C), using 40ug of isolated genomic dna as template per sample, following this protocol, https://media.addgene.org/cms/filer_public/61/16/611619f4-0926-4a07-b5c7-e286a8ecf7f5/broadgpp-sequencing-protocol.pdf. PCR amplicons were agarose gel purified using nucleoSpin Gel and PCR Clean‑up Mini kit (Machery-Nagel cat 740609.50)
CRISPR sgRNA amplicons
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Fastq files were aligned to Calabrese (Addgene #92379, #92380) or Dolcetto (Addgene #92385, #92386) libraries using MAGeCK version 0.5.9.2 count command using --trim-5 22,23,24,25,26,28,29,30 argument
Genome_build: NA
Supplementary_files_format_and_content: Raw counts of sgRNAs
|
| |
|
| Submission date |
May 11, 2021 |
| Last update date |
May 13, 2021 |
| Contact name |
Zachary Steinhart |
| E-mail(s) |
[email protected]
|
| Organization name |
J. David Gladstone Institutes
|
| Department |
Gladstone-UCSF Institute of Genomic Immunology
|
| Lab |
Marson Lab
|
| Street address |
1650 Owens St
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE174255 |
CRISPR activation and interference screens decode stimulation responses in primary human T cells [CRISPR] |
| GSE174292 |
CRISPR activation and interference screens decode stimulation responses in primary human T cells |
|
| Relations |
| BioSample |
SAMN19108255 |
| SRA |
SRX10856282 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
