2025-03-20
ENA first public
ERC000011
ENA-CHECKLIST
The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology
INSDC center name
public
INSDC status
ERS23780734
SRA accession
E-MTAB-14909:off -target  site3_S41_2
Submitter Id
ArrayExpress
broker name
293T/17
cell_line
epithelial
cell_type
not collected
collection date
human
common name
Protocols: Transfected cells were harvested two to three days after Cas9 RNP transfection. To detach and dissociate the cells, they were treated with trypsin. Cell pellets were then collected by centrifugation. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (cat# 69506). The concentration of genomic DNA ranges from 100 to 200 ng/μl.   The edited regions in target genes were amplified by PCR and sequenced on an Illumina MiSeq. Amplification primers containing partial Illumina forward and reverse adapters were used in the first round of PCR to amplify the edited regions. The first PCR reaction (25 µl) contained 12.5 µl of Q5 High-Fidelity 2X Master Mix (cat# M0492L), 0.5 µM forward and reverse primers, and 150 ng of genomic DNA. The PCR program was as follows: 98°C for 3 minutes; 30 cycles of 98°C for 15 seconds, optimized annealing temperature for 30 seconds, and 72°C for 15 seconds; followed by a final extension at 72°C for 2 minutes. PCR products were analyzed by gel electrophoresis, and concentrations were quantified. In the second PCR, full-length Illumina adapters were added to each sample using NEBNext Multiplex Oligos for Illumina (NEB, cat# E6440S). The second PCR reaction (50 µl) contained 25 µl of Q5 High-Fidelity 2X Master Mix, 0.125 µM index primers (higher concentrations resulted in amplification bias), and 1 ng of first-round PCR products. The program for the second PCR was as follows: 98°C for 1.5 minutes; 12 cycles of 98°C for 15 seconds, 72°C for 30 seconds, and 72°C for 20 seconds; followed by a final extension at 72°C for 2 minutes. PCR products were analyzed by gel electrophoresis and purified using the NucleoSpin Gel and PCR Clean-Up Kit. DNA concentration was measured using Qubit. All samples were pooled at an equimolar ratio and sequenced on the Illumina MiSeq platform.
description
off-target site 3
genetic modification
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
kidney
organism part
Homo sapiens
scientific_name
off -target  site3_S41_2
title