GSM9248863: Sort-seq, 3SS and branch site stem loop library, bin 6; Saccharomyces cerevisiae; OTHER
Yeast cells were harvested and resuspended at low density in H2O. Cell walls were digested in 20 µl reactions with 1 µl Zymolase (Zymo Research) in 25 mM sodium phosphate pH 7.5 for 20 min at room temperature, then 5 min at 37 ºC. Samples were diluted with 60 µl H2O and 2 µl of the resulting crude gDNA was used as template for amplicon sequencing. the variable region was amplified by PCR with 15 cycles using KAPA HiFi HotStart polymerase (Roche) with primers adding Illumina flowcell binding sequences. Reactions were purified using AMPure XP beads (Beckman) and used as template for a second round of PCR with Illumina TruSeq i5 and i7 index primers for 15 cycles. Final libraries were again purified with AMPure XP beads, pooled and sequenced on Illumina NovaSeq machines in 2x150 paired-end mode.