Cells were seeded at the amount of 1 cell per well in a 96-well plate by FACS (S3e Cell Sorter, Bio-Rad) 48 h after transfection. After reaching a ∼80–90% confluency, cells were divided into two equal portions and seeded in two 96-well plates. One of the plates was used for mutation screening by T7 endonuclease I (T7EI) cleavage assay (Kim et al., 2009 (link)). Genomic DNA was isolated using genomic DNA Isolation Kit (BIOLABMIX Ltd., Novosibirsk, Russia), PCR was performed using specific primers (5’-AGCCTTTGTCTGCTAAGGTCA-3’ and 5’-GTTGCCATTAACCGATGTCGA-3’ for SNORD74, 5’-TGGTATGTTACCTGCATCATTGG-3’ and 5’-TAGGTGTACTCTCTATGTTCCC-3’ for SNORD75, 5’-GAGTGCTAGAATGATGAGG-3’ and 5’-TCCAGCTTTCTGTCTAATGCC-3’ for SNORD77, 5’-ATTACAGGCATGTGACACC-3’ and 5’-CACTCCCATCTACAGATTAAGG-3’ for SNORD80), and the amplification products were annealed and subjected to T7EI (NEB) according to the manufacturer’s protocol. Mutations were identified by TA-cloning of the PCR products using CloneJET Kit (Thermo Fisher Scientific) and E. coli strain XL1-Blue followed by Sanger sequencing with further analysis of the obtained data by Tracking of Indels by Decomposition (TIDE) assay (Brinkman et al., 2014 (link)).
Free full text: Click here
