 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 24, 2024 |
| Title |
Donor 2 Treg, Resting, CD28-Low |
| Sample type |
SRA |
| |
|
| Source name |
Regulatory T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Regulatory T cells
treatment: Resting
|
| Treatment protocol |
Cells were harvested at rest or restimulated for the indicated length of time with 1uL/mL Cell Activation Cocktail without Brefeldin A (Biolegend #423302).
|
| Growth protocol |
All cells were cultured in Complete X-VIVO [cX-VIVO: Lonza Bioscience #04-418Q, 5% FCS (R&D Systems, lot #M19187), 55uM 2-mercaptoethanol, and 4mM N-Acetyl L-Cysteine]. Regulatory and Conventional T cells were activated by CTS Dynabeads Treg Xpander (Thermo Fisher #46000D) or CTS Dynabeads CD3/CD28 (Thermo Fisher #40203D), respectively, with 1:1 cell:bead ratio at 1e6 cells/mL in cX-VIVO supplemented with recombinant human IL-2. Primary human Tconv cells were activated and maintained in 300U/mL rhIL-2. Treg cells were activated in 300U/mL rhIL-2 and subsequently maintained in 200U/ml rhIL-2.
|
| Extracted molecule |
other |
| Extraction protocol |
Sorted samples were pelleted and resuspended in 400uL ChIP Lysis Buffer (1% SDS, 50mM Tris, pH 8, 10mM EDTA) per 5e6 cells. Each 400uL reaction received 16µl NaCl (5M) and was incubated at 66°C overnight. Subsequently, each reaction received 8µl RNAse A (Fisher Scientific #EN0531) and was incubated at 37°C 1h. Then, 8µl Proteinase K (Fisher Scientific #25530049) was added and the samples incubated at 55°C 1h. One phase lock tube (Quantabio, #2302820) per sample was spun at 20,000g 1 minute and received 400µl Phenol:Chloroform:Isoamyl Alcohol (25:24:1). 400µl sample was added to each phase lock tube, shaken vigorously, and centrifuged at 20,000g 25°C 5 minutes. Aqueous phases were transferred to low-binding tubes (Eppendorf, #022431021) and received 40µl of Sodium Acetate (Fisher Scientific #46-033-CI), 1µl GlycoBlue (Invitrogen, #AM9515), and 600µl isopropanol. Samples were vortexed and frozen at -80°C ≥30 minutes. Frozen samples were centrifuged 20,000g 4°C 30 minutes, pellets washed with fresh 70% ethanol, and allowed to air dry for 15 minutes. Genomic DNA pellets were resuspended in Zymo DNA elution buffer (Zymo, #D3004-4-10) and reconstituted at 65°C for 1 hour, or until dissolution.
Sequencing libraries were generated using 3.75ug genomic DNA per 50uL PCR reaction with 0.25uM CM_oligo_4 and 0.25uM unique p7 reverse primer as in CM_oligo_5 (see Supplementary Information). PCR reactions were run with the following parameters: 95°C 1’, [95°C 30”, 60°C 30”, 72°C 30”] x 28, 72°C 10’, 4°C hold. Amplicons were purified with DNA Clean & Concentrator-25 kits (Zymo Research #D4033)100. One sample (Donor 2 Tconv cells, ICOS screen) was re-indexed before sequencing. Pooled libraries were sequenced with a custom sequencing primer CM_oligo_6 on an Illumina NextSeq500 instrument.
|
| |
|
| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
sgRNA library sequencing
|
| Data processing |
Raw Illumina sequencing data were demultiplexed and fastqs generated using bcl2fastq (v2.20.0).
Short guide RNA abundances were quantified using MAGeCK (v0.5.9.4) with a reference file listing sgRNA sequences, a sgRNA ID, and the 5’ genomic position of the sgRNA (hg38).
Unnormalized sgRNA count files for each sample were loaded into R (v4.1.2), and statistical testing of sgRNA effects across donors was performed with DESeq2 (v1.34.0).
Assembly: hg38
Supplementary files format and content: .rds file containing differential sgRNA abundance in High vs Low populations across all samples as quantified by DESeq2
Library strategy: sgRNA Amplicon
|
| |
|
| Submission date |
Mar 11, 2024 |
| Last update date |
Mar 24, 2024 |
| Contact name |
Alexander Marson |
| Organization name |
Gladstone-UCSF Institute of Genomic Immunology
|
| Street address |
1650 Owens Street
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE261330 |
Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells [CRISPRi] |
| GSE261332 |
Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells |
|
| Relations |
| BioSample |
SAMN40378701 |
| SRA |
SRX23901379 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
