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| Status |
Public on Apr 26, 2021 |
| Title |
Donor_1_CBFB_RNA |
| 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 isolated from 3 donors using consented Luekopaks (STEMCELL). Guide RNAs against 24 targets or 8 AAVS1 controls were assembled into Cas9 ribonucleoproteins and electroporated into the cells. Five days after electroporation cells were collected.
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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 |
total RNA |
| Extraction protocol |
Cells were lysed with Zymo QuickRNA lysis buffer and isolated with the Zymo QuickRNA micro kit (Zymo, Cat #R1050) using the manufacturer's protocol. RNA samples were treated with 1.5 ul of Turbo DNase (Invitrogen, Cat # AM2238) and then cleaned up using the Zymo RNA-5 Clean and Concentrator (Zymo, Cat #R1016). Isolated RNA integrity and concentration was checked using Agilent RNA Screen Tapes (Agilent, Cat #5067-5576)
RNA was submitted to the UC Davis DNA Technologies and Expression Analysis Core to generate 3’ Tag-Seq libraries with unique molecular indices (UMIs). Barcoded sequencing libraries were prepared using the QuantSeq FWD kit (Lexogen, Vienna, Austria) for multiplexed sequencing according to the recommendations of the manufacturer (Lexogen). The fragment size distribution of the libraries was verified via micro-capillary gel electrophoresis on a Bioanalyzer 2100 (Agilent, Santa Clara, CA). The libraries were quantified by fluorometry on a Qubit fluorometer (LifeTechnologies, Carlsbad, CA), and pooled in equimolar ratios. Samples were sequenced on a HiSeq 4000 sequencer (Illumina, San Diego, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RNA_UMI_dedup_counts.txt
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| Data processing |
Adapters were trimmed from fastq files using cutadapt version 2.10 with default settings keeping a minimum read length of 20 bp. Reads were mapped to the human genome GRCh38 keeping only uniquely mapping reads using STAR version 2.7.b with the following settings “--outFilterMultimapNmax 1”. UMIs were extracted from fastqs using umi_tools version 1.0.1 extract command with the following settings “--extract-method=regex --bc-pattern='(?P<umi_1>.{{6}})(?P<discard_1>.{{4}}).*'”. Reads were then deduplicated using umi_tools dedup command with default settings. Deduplicated reads overlapping genes were then counted using featureCounts version 2.0.1 with the following settings “-s 1” and using the Genocde version 35 basic transcriptome annotation.
Genome_build: hg38
Supplementary_files_format_and_content: UMI deduplicated RNA-Seq 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) |
| GSE171677 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [RNA-Seq] |
| GSE171737 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks |
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| Relations |
| BioSample |
SAMN18672163 |
| SRA |
SRX10549806 |
| 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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