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| Status |
Public on Nov 25, 2021 |
| Title |
Amplicon_combo_ko_Donor1_gDNA7_STAT5B |
| 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 |
GuideRNA/Cas9 ribonucleoprotein complexes were electroporated into CD4+CD25- T 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 |
On day 5 post-electroporation, genomic DNA was isolated from each sample using DNA QuickExtract (Lucigen, Cat #QE09050) according to the manufacturer’s protocol.
Primers were designed to flank each sgRNA genomic target site. Amplicons containing CRISPR edit sites were generated by adding 1.25 µL each of forward and reverse primer at 10nM to 5 µL of sample in QuickExtract, 12.5 µL of NEBNext Ultra II Q5 master mix (NEB, Cat #M0544L), and H2O to a total 25 µL reaction volume. The following PCR cycling conditions were used: 98°C for 3 minutes, 15 cycles of 94°C for 20 seconds followed by 65°C-57.5°C for 20 seconds (0.5°C incremental decreases per cycle), and 72°C for 1 minute, and a subsequent 20 cycles at 94°C for 20 seconds, 58°C for 20 seconds and 72°C for 1 minute, and a final 10 minute extension at 72°C. Samples were then diluted 1:200 and subsequently Illumina sequencing adapters and indices were added in a second PCR reaction. Indexing reactions included 1 µL of the diluted PCR1 sample, 2.5 µL of each the forward and reverse indexing primers at 10 µM each, 12.5 µL of NEB Q5 master mix, and H2O to a total 25 µL reaction volume. The following PCR cycling conditions were used: 98°C for 30 seconds, followed by 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds for 12 cycles, and a final extension period at 72°C for 2 minutes. Samples were quantified in a 96-well plate reader using the Quant-IT DNA high sensitivity assay kit (Invitrogen, Cat #Q33232) according to the manufacturer’s protocol and pooled into 1 tube. Post pooling, samples were then SPRI purified, and quantified using an Agilent 4200 TapeStation. Samples were then sequenced on an Illumina NovaSeq with PE 300 reads.
Amplicon sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Adapter sequences were trimmed from fastq files using cutadapt version 2.8 using default settings keeping a minimum read length of 50 bp. Insertions and deletions at each CRISPR target site were then calculated using Crispresso2 version 2.0.42 (Clement et al. 2019) with the following options "--quantification_window_size 3" and "--ignore_substitutions".
Genome_build: hg38
Supplementary_files_format_and_content: Combo_KO_amplicons_modified.txt
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| Submission date |
Nov 24, 2021 |
| Last update date |
Nov 25, 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 |
GPL24676 |
| Series (2) |
| GSE171737 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks |
| GSE189523 |
Systematic discovery and perturbation of regulatory genes in human T cells reveals the architecture of immune networks [Amplicon III] |
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| Relations |
| BioSample |
SAMN23427416 |
| SRA |
SRX13219773 |
| 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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