Single-cell whole-genome sequencing of an isogenic Barrett's esophagus cell line model of varying chromosomal instability
Shallow single-cell whole-genome sequencing (scWGS) was performed on isogenic non-dysplastic Barrett's esophagus CP-A (KR-42421) cell lines exhibiting distinct inherent levels of chromosomal instability (CIN). CIN was progressively induced through sequential CRISPR-Cas9-mediated depletion of TP53 (p53) and CDKN2A (p16), followed by overexpression of a dominant-negative form of the mitotic kinesin MCAK (dnMCAK). Serial genetic alterations were introduced independently across three clonal lineages. A Cas9-expressing parental CP-A clone was included as a control to account for potential confounding effects of Cas9 overexpression.Sequencing was performed at the Research Sequencing and iPSC/CRISPR Facility, ERIBA, University Medical Centre Groningen, University of Groningen.Isolation and staining of nuclei, as well as nuclei sorting and library preparation were performed as previously described (van den Bos et al., 2016), with small variations. Single nuclei for each cell line were dry sorted into 96-well plates. Overnight re-hydration in TPLK was performed and proteolysis started. MNase Digestion, AMPure Bead Cleanup and End Repair and A-Tailing steps were replaced in favor of the Fragmentase enzyme (NEBNext Ultra II FS DNA Library Prep Kit, New England Biolabs). Subsequent library preparation steps were unchanged.Libraries were sequenced on a NextSeq2000 (Illumina) P1-100c (1M reads per single cell) sequencer, using a 77bp read-length and 11bp dual index read configuration. Resulting FASTQ files were mapped to the human reference genome GRCh38 using the Burrows-Wheeler aligner. The aligned read data (BAM files) were analyzed using the AneuFinder algorithm (Bakker et al., 2016).