GSM7990653: experiment 1 replicate 1, scRNAseq; Saccharomyces cerevisiae; Candida albicans; RNA-Seq
At the time of sampling, 3 mL of yeast cultures were immediately spun down in a room temperature centrifuge at 3000G for 5 minutes. The media supernatant was removed, and the cell pellet was resuspended in 2 mL of cold 4% formaldehyde in molecular grade PBS. The cells were then fixed cold in a 4C refrigerator for approximately 18 hours. After fixation, the samples were spun down in a 4C centrifuge at 3000G for 5 minutes. The formaldehyde supernatant was removed, and the pellet was resuspended in 1 mL cold molecular grade 100 mM Tris HCl pH 7 plus 0.1 U/uL SUPERase-In RNase inhibitor (Invitrogen) to quench the formaldehyde. Cells were then centrifuged, resuspended in 250 uL of a zymolyase enzymatic solution of 0.1M Na2EDTA, 1M sorbitol, and 0.005 U/uL zymolyase (Zymo Research) (pH ~7.5), and incubated at 37C for 15 minutes. At the end of the incubation, 1 mL of cold PBS plus 0.1 U/uL SUPERase-In RNase inhibitor and 0.1 U/uL Enzymatics RNase inhibitor (Enzymatics) was immediately added, and the cells were centrifuged at 4C, 3000G for 5 minutes. The cells were then resuspended in 250 uL of cold 0.4% Triton X-100 in PBS plus RNase inhibitors and incubated on ice for 3 minutes. Again, 1 mL of cold PBS plus RNase inhibitors was added at the end of the 3 minutes and the cells were centrifuged. They were then resuspended in 500 uL of cold PBS plus RNase inhibitors, vortexed on high for ~30 seconds, and lightly filtered through a 40 µm pluriStrainer (pluriSelect). The cells were then counted using a Beckman Coulter Cell Counter and diluted to 1 million cells/mL into fresh cold PBS plus RNase inhibitors. The cells were then processed according to the standard SPLiTseq protocol. Briefly, in situ reverse transcription and ligation steps were performed and then the cells were lysed and cDNA collected and amplified for sequencing.