GSM7900889: WT yeast, 10 minute 4-TU treatment; Saccharomyces cerevisiae; RNA-Seq
Fixed cells were incubated on ice for 15min, then pelleted and washed twice with ice cold PBS + 0.01% BSA. Cells were resuspended in 400ul PBS + 0.01% BSA, and the cell suspension was added to 1mL of alkylation buffer, followed by a 45min incubation at 45°C with occasional inversion. Cells were pelleted and washed twice with PBS + 0.01% BSA + 10mM DTT, then washed once with 1M sorbitol + 0.01% BSA. Cells were then resuspended in 900ul spheroplasting buffer, and 100ul of 100mg/ml Zymolyase 100T was added. Cells were spheroplasted for ~30min at 30°C with occasional inversion. The spheroplasting mixture was filtered through a MACS SmartStrainer, and spheroplasts were pelleted then washed twice in 1M sorbitol + 0.01% BSA. Spheroplasts were counted immediately prior to GEM generation using a hemocytometer. The Chromium Next GEM Single Cell 3ʹ Kit (10x Genomics, V3.1) protocol was performed according to manufacturer's instructions. Briefly, GEMs containing Gel Beads decorated with poly(dT) capture sequences (including Illumina TruSeq Read 1 sequence, 10x Cell barcode, and UMI) were prepared. GEMs were combined with prepared yeast spheroplasts, resulting in lysis of the spheroplasts in droplets and capture of PolyA-containing RNA on Gel Beads. Captured RNAs were reverse transcribed, GEMs were broken and pooled, and cDNA was amplified resulting in the installation of i5 and i7 sample indexes. Prepared paired-end libraries were sequenced on an Illumina NextSeq 2000 instrument using P3 reagents, with read configuration of 28nt for R1 to identify cell barcode and UMIs, and 125nt for R2 to sequence the captured mRNA.