title: Amplicon-Screening of exonic RNA 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 RNA 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. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, 74106) and reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen, 11754250). For cDNA sequencing of mutant cell libraries, RNA was reverse transcribed and amplified in 5 reactions with 1 µg RNA template using SuperScript IV One-Step RT-PCR System with ezDNase (Invitrogen, 12595025). The amplified PCR products were pooled, providing an estimated variant coverage of 50-250x, purified and diluted 1:1000 for a second step of PCR, using exon-specific primers and Q5 High-Fidelity DNA Polymerase.