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Sample GSM5732836

Query DataSets for GSM5732836

Status

Public on Feb 04, 2022

Title

Donor15_IFNG_high_Rep1

Sample type

SRA

Source name

Primary CD4+ T cells

Organism

Homo sapiens

Characteristics

donor: donor 15
group: IFNG_high

Treatment protocol

One day after activation, T cells were infected with dCas9-VP64 (CRISPRa) lentivirus. Two days after activation, T cells were infected with Calabrese Set A (addgene 92379) and Calabrese Set B (addgene 92380) lentivirus (mixed 50/50) 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, IFN-γ low and high, or TNF-a low and high bins.

Growth protocol

Primary human CD4+ 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)

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

other (CRISPR sgRNA capture)

Data processing

Library strategy: CRISPR sgRNA amplicons
Fastq files were aligned to Calabrese (Addgene #92379, #92380) 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

Dec 13, 2021

Last update date

Feb 04, 2022

Contact name

Zachary Steinhart

E-mail(s)

zachary.steinhart@gladstone.ucsf.edu

Organization name

J. David Gladstone Institutes

Department

Gladstone-UCSF Institute of Genomic Immunology