Computational protocol: ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability

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Protocol publication

[…] The ID3 CRISPR (Guide sequence insert: 5ʹ-CGAGGCGGTGTGCTGCCTGTCGG)-3ʹ construct targeting exon 1 of the human ID3 gene were designed using the Cas9-Designer (http://www.rgenome.net/cas-designer). SpCas9 expression plasmids (500 ng) and sgRNA plasmids (500 ng) were transfected to 2 × 105 U2OS cells using 4D-nucleofector (Lonza) SE kit and program CM-104 in 20 µl Nucleovette Strips. Genomic DNA was isolated with the Nucleospin Tissue Kit (MACHEREY-NAGEL) 72 h post-transfection. Target sites were amplified with adapter primers using Phusion polymerase (New England Biolabs). The resulting deep sequencing libraries were subjected to paired-end sequencing with the MiSeq system (Illumina). After MiSeq, paired-end reads were joined by the Fastq-join. Fastq-joined files were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/) to obtain a mutation frequency in edited cells. For single-cell colony expansion, transfected cells were diluted and sorted with a density of 1 cell per well into 96-well-plates. After 7–10 days incubation, only single-cell colonies were screened visually under microscope. When colonies reached 70% confluence, the cells were transferred to 24-well-plates. Genomic DNA was isolated from each single colony. Sequences of ID3 target region were analyzed by a targeted deep sequencing in each single-cell colony and further confirmed by Sanger sequencing. […]

Pipeline specifications

Software tools Cas-Designer, ea-utils, Cas-Analyzer
Application Genome edition