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Sample GSM8125122 Query DataSets for GSM8125122
Status Public on Mar 05, 2024
Title CD55_parental-gCH27
Sample type SRA
 
Source name K562
Organism Homo sapiens
Characteristics cell line: K562
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.
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).
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
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina MiSeq
 
Description gCH27 = crCD55-4
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
 
Submission date Mar 04, 2024
Last update date Mar 05, 2024
Contact name Chris Hsiung
Organization name Arc Institute
Lab Luke Gilbert Lab
Street address 3181 Porter Dr
City Palo Alto
State/province CA
ZIP/Postal code 94304
Country USA
 
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
Relations
BioSample SAMN40253978
SRA SRX23829838

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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