RNA-seq of yeast with human chr7 YACs

RNA-seq was performed of S. cerevisiae harboring human chr7 YACs in order to gauge the frequency and form of transcription in naïve DNA (i.e. DNA that did not evolve in the host cell) The YACs HSC7E1068 and HSC7E481 — originally constructed by the Scherer lab as part of the human chromosome 7 specific YAC library — was obtained from the Centre for Applied Genomics (TCAG) (Toronto, Ontario) as a yeast extract peptone dextrose (YPD) stab. Based on the RNA-seq data, the YACs comprised from 80,372,600 to 81,132,754 (HSC7E1068) and 87,346,480 to 88,157,340 (HSC7E481) of human chromosome 7 (hg38), which was consistent with the previously mapped locations. To select for yeast containing the YACs, yeast were streaked onto synthetic defined (SD) -URA plates containing adenine sulfate (Sunrise Science, USA) and were grown at 30 °C for two days. Colony PCR was performed on red colonies to confirm the presence of the YAC1068 and YAC481 with primer sets E1068F (5’-CGCTGAGGACAACACAGTCT-3’), E1068R (5’-ACACTGTCTGGAAAGCTCGG-3’) and E481F (5’-GCTCATAGCATGGCTTGTGC-3’), E481R (5’-CCCACTCCTACTGTAGCCCT-3’) respectively, using a protocol described previously (73). Primers were designed to amplify a portion of CD36 and ABCB1 - genes reported to be found in YAC1068 and YAC481 respectively (74), which was later confirmed by our RNA-seq results. Yeast colonies containing either YAC1068 or YAC481 were grown in liquid SD-URA to an OD600 of ~0.3 - 0.6. Total RNA was extracted from liquid yeast cultures using the hot acid phenol method, described previously (75). Genomic DNA was removed from RNA using Turbo DNase following the manufacturer's instructions (Invitrogen). Extracted RNA was prepared for strand specific sequencing by the Biomedical Research Centre Sequencing Core (UBC, Vancouver) using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina and polyA selection (NEB). Libraries were sequenced on an Illumina Miseq machine using 300 cycles, generating 20 million paired end reads.