title: Amplicon-Screening of exonic DNA regions in exon-wide mutant cell libraries of mutant TP53 (Exons 5, 6, 7, 8)  in HCT116 cell line clones generated via CRISPR editing.
design: Amplicon-Screening of exonic DNA regions in exon-wide mutant cell libraries of mutant TP53 (Exons 5, 6, 7, 8)  in HCT116 cell line clones generated via CRISPR editing.
library_construction_protocol: For generating exon-wide mutant cell libraries, 5.4 x 108 HCT116Δin5/LSL or HCT116Δin5+7/LSL cells were transfected with 1.125 mg pX459_blast_In5LSL or pX459_blast_In7LSL, and 1.125 mg of the corresponding HR700 vector library using Lipofectamine 2000 according to the manufacturer's protocol. Transfected cells were selected with 20 µg/ml blasticidin for 3 days and 1 µg/ml puromycin for 7 days. 1.2 x 108 cells were infected with AV-Cre.  The human colorectal carcinoma cell line HCT116 was obtained from the American Tissue Collection Center (ATCC, CCL-247) and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, 41966-029) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, S0615) and 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco, 15140122). To ensure unambigous genotype-phenotype correlations, the cell were haploidized prior to CRISPR-editing by introducing inactivating deletions of intronic splicing into one of the two TP53 alleles, leaving only one functional copy of TP53. The remaining TP53 allele was altered by inserting a LoxP-flanked transcriptional stop cassette (Lox-Stop-Lox, LSL) into intron 4, which allowed reversible silencing of TP53 expression. The LSL cassette was then specifically targeted with CRISPR/Cas9 to introduce a variety of different mutant p53 alleles. After 5 days, the cell library was divided and treated with 10 µM N3a or DMSO for 8 days. Genomic DNA of mutant cells was isolated using DNA Blood Mini Kit (QIAGEN, 51106) following the manufacturer's protocol. A nested PCR strategy was used to selectively amplify either edited or edited and recombined alleles (Extended Data Fig. 1a). The input amount of genomic DNA and number of PCR reactions were adjusted to achieve a minimum average coverage of 1000 cells/variant. After the first PCR step, The PCR products were pooled, purified and diluted 1:1000 for the second step. Editing-specific primers were used for the second step PCR, exon-wise. The PCRs were performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs, M0491) following the manufacturer's protocol. PCR products were purified using PCR purification kit according to manufacturer's protocol. PCR amplicons were purified with AMPure XP beads (Beckman Coulter, A63880) and sequencing libraries were prepared from 5 ng of the purified amplicon using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, E7370L) according to manufacturer's protocol.