DNAJB6 variant screen in yeast
Genomic DNA was extracted from the yeast culture using a lithium acetate-sodium dodecyl sulphate (LiOAc-SDS) lysis method. Following DNA extraction, sequencing library preparation was conducted with two sequential rounds of PCR. First, the barcodes associated with each DNAJB6 variant were amplified in 16 independent reactions per sample, appending universal Illumina adapters to the 5 and 3 ends of the amplicon. Replicate first-round PCR products were pooled and subjected to a second round of PCR in which unique indices were appended for each sample in technical duplicate. All second-round PCR products were then gel purified, pooled, and sequenced on an Illumina NextSeq 2000 with 100bp single-end reads.