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| Status |
Public on Mar 05, 2024 |
| Title |
CD81_parental-gCH29 |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
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| Treatment protocol |
K562 cell lines engineered with the corresponding Cas12a protein constructs were transduced with crRNAs, sorted for transduced cells based on GFP-positivity. 200,000 cells were collected on day 14 after crRNA transduction.
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| Growth protocol |
K562 cells were cultured at 37deg. C with 5% CO2 in tissue culture incubators. Culture media consists of RPMI-1640 (Gibco cat# 22400121) containing 25 mM HEPES, 2mM L-glutamine, and supplemented with 10% FBS (VWR), 100 units/mL streptomycin, and 100 mg/mL penicillin. During the screen, K562 cells were cultured in flasks in a shaking incubator and the culture media was supplemented with 0.1% Pluronic F-127 (Thermo Fisher P6866).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For analysis at the CD55 and CD81 loci, genomic DNA was isolated using NucleoSpin Blood (Macherey-Nagel, Catalog no. 740951.50). For analysis at the KIT locus, cell lystates were generated using QuickExtract DNA Solution (Lucigen) and directly subjected to PCR amplification.
For analysis of CD55 and CD81 loci, PCRs for loci of interest were run using Amplicon-EZ (Genewiz) partial Illumina adapters and amplicons were processed using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Catalog no. 740609.250). Paired end (2 x 250 bp) sequencing was completed at GENEWIZ (Azenta Life Sciences). For analysis of the KIT locus, cell lysates were subjected to 15 cycles of PCR to introduce Illumina sequencing primer binding sites and 0-8 staggered bases to ensure library diversity. After reaction clean-up using ExoSAP-IT kit (Thermo Fisher 78201), an additional 15 cycles of PCR was used to introduce unique dual indices and Illumina P5 and P7 adaptors. Libraries were pooled and purified by SPRIselect magnetic beads before paired-end sequencing using an Illumina MiSeq. Sequencing primer binding sites, unique dual indices (from Illumina TruSeq kits), P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document # 1000000002694 v16.
PCR amplicon sequencing
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
gCH29 = crCD81-1
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| Data processing |
Raw fastq files were aligned to reference sequences using CRISPResso2, as detailed in the associated manuscript.
Assembly: hg19
Supplementary files format and content: Quantification of percentage of reads containing indels overlapping each position in the PCR amplicon
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| Submission date |
Mar 04, 2024 |
| Last update date |
Mar 05, 2024 |
| Contact name |
Chris Hsiung |
| Organization name |
Arc Institute
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| Lab |
Luke Gilbert Lab
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| Street address |
3181 Porter Dr
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| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94304 |
| Country |
USA |
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| Platform ID |
GPL15520 |
| Series (2) |
| GSE260830 |
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (amplicon) |
| GSE260832 |
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations |
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| Relations |
| BioSample |
SAMN40253977 |
| SRA |
SRX23829839 |
| 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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