Dissecting the yeast transcriptome landscape through genetic and environmental Perturb-seq analyses
Dissecting the yeast transcriptome landscape through genetic and environmental Perturb-seq analyses
Conditions and sample collection: We subjected or not the cell pool to osmotic stress 0.4M NaCl for 15 minutes and followed the cell fixation protocol from GEXSCOPE® Microbial Single Cell RNA Library Kit HD (Yeast) (4161031). The selection of this experimental conditions is based on a combination of extensive transcriptomics data that determines the peak of expression of osmoresponsive genes. Briefly, 100 ml cells where centrifuged 1 min 3000 RPM, media poured off and cells immediately fixed and resuspended in 10 ml of ice cold 80% Methanol (Scharlab, ME0301005P). For each condition methanol-fixed cells were split into 20 different aliquots (1 ml) from each condition (control/NaCl) and stored at -20ºC, to avoid multiple freezing and thawing.  Growth: Frozen glycerol stocks from the transcriptionally barcoded yeast knock out collection were recovered on URA- media at 25ºC for 48 hours until saturation. The next day 5 µl of cultures were seeded into a new 96-well plate containing 200 µl of YPD at an average optical density of XX# measured with Microplate Reader Sinergy HX1. Cells were allowed to recover for 6 hours until they reached mid-exponential phase average OD660, pooled together by pouring the media into a large flask and shacked vigorously to ensure a homogeneous mixture. To maximize the representativeness of each mutation optical density for all plates was assessed after seeding and before pooling.  Conditions: We subjected or not the cell pool to osmotic stress 0.4M NaCl for 15 minutes and followed the cell fixation protocol from GEXSCOPE® Microbial Single Cell RNA Library Kit HD (Yeast) (4161031). The selection of this experimental conditions is based on a combination of extensive transcriptomics data that determines the peak of expression of osmoresponsive genes. Briefly, 100 ml cells where centrifuged 1 min 3000 RPM, media poured off and cells immediately fixed and resuspended in 10 ml of ice cold 80% Methanol (Scharlab, ME0301005P). For each condition methanol-fixed cells were split into 20 different aliquots (1 ml) from each condition (control/NaCl) and stored at -20ºC, to avoid multiple freezing and thawing. Cells were lysed within the GENEXscope singleron matrix. Lysis buffer was done following manufacturer's instructions.  Transcriptome Library Preparation: To perform the yeast Perturb-seq experiment we followed GEXSCOPE® Microbial Single Cell RNA Library Kit HD instructions. Briefly, 1 aliquot of each condition was equilibrated for 15 minutes at 4ºC (ice) before being pelleted (2 min 1640 g at 4ºC). Supernatant was discarded and cells were washed twice with 700 µl of rehydration buffer (DPBS1X, BSA 20 mg/ml (Thermo Fisher Scientific, 14190144 and AM2616), RNASe inhibitor (40U/µl) (Takara, 2313A), Actinomycin D (2mg/ml) (Sigma, A1410-2MG)). To disaggregate potential cell clumps we filtered twice with PluriStainer (PluriSelect, 43-50040-03) and cells were counted with a Neubauer chamber. A total of 210,000 cells (120 µl) of the yeast suspension was loaded into a High Density Singleron Matrix v1.0.1instrument. For each run, a total of 2 matrix-cartridges were run simultaneously each one loaded with control and NaCl simultaneously. The position of each sample in the Singleron Matrix instrument (upper/lower slot) was exchanged per every run. A total of 29 matrix-cartridges were run (14 control and 15 NaCl). Single-Yeast Partitioning, mRNA capture, reverse transcription, cDNA amplification and cDNA purification were performed according to manufacturer's instructions. For cDNA library amplification 8 PCR cycles were used. Samples were processed per run (as group of two samples, control/NaCl) until cDNA purification. The cDNA quality of each cDNA was assessed by Qubit (ThermoFisher Scientific, Qubit™ 1X dsDNA HS Assay Kits, Q33231), the size and integrity of the full-length cDNA was inspected with a DNA Pico Bioanalyzer chip (Agilent technologies). Library preparation was done using 50 ng of purified cDNA as an input and library amplification was done with 10 PCR cycles. Finally, library quality check was performed  Equimolar pools of the 29 libraries were pooled together and sequenced in one S4 NovaSeq lane (CeGAT, Tübingen) using paired end 150 cycles (PEx150 cycles).  Targeted amplification libraries (TA): For each full length cDNA library, 3 ng were used as a template for targeted amplification of the URA3 transcript was achieved by a single step PCR with indexed primers. PCR products of targeted amplification were purified by Ampure beads (0.8X). Library quality and molecular weight were assessed by Bioanalyzer. Equimolar ratios all all 29 libraries were pooled together and sequenced paired end 150 bp using an Illumina NextSeq500.