title: Illumina MiSeq sequencing; Uncovering novel genetic factors controlling sphingolipid-induced mitochondrial cell death in yeast through genome-wide transposon-insertion mutant screening (SATAY)
design: Uncovering novel genetic factors controlling sphingolipid-induced mitochondrial cell death in yeast through genome-wide transposon-insertion mutant screening (SATAY)
library_construction_protocol: After the respective treatment, cells were collected by centrifugation, washed with ice-cold water, and frozen as 500mg aliquots at -80ºC. SATAY was performed as described Michel, A.H and  Kornmann, B. (2022 Yeast Functional Genomics. Methods in Molecular Biology, vol 2477), with some modifications. ByK352 hfd1∆::KanMX strain was transformed with pBK549 plasmid containing the transposon, obtained from the Kornmann Lab (Michel & Kornmann, 2022). Transformed cells were inoculated into 200mL of SC liquid + 0.2% Glucose + 2% Raffinose -Uracil at OD600 of 0.15 and the culture was grown until saturation (OD600 3.9) for 16 hours. Then, the culture was concentrated by centrifugation to obtain a final OD600 of 39. From this culture, 200µL were plated on 50x8.5cm plates containing 25mL of SC + 2% Galactose -Adenine using glass beads. Plates were incubated for 3 weeks at 30ºC. After incubation clones reached a density on the plate of 46750 colonies/plate, on average, with a total number of 2.3E+06 generated mutants. All colonies were then scrapped with very low volume of sterile water and pooled together.  From the pool, two independent cultures of 1L were inoculated in SC +  2%Glucose -Adenine. The control one was inoculated at an initial OD600 of 0.2 and grown until saturation (OD600 of 8) at 30ºC for 36 hours. The other culture, used for t-2-hex treatment, was grown for 36 hours from an initial OD600 of 0.05 to 3, and then diluted to OD600 0.1 in 500mL of SC +2% Glucose -Adenine treated with 50µM of t-2-hex (Avanti Polar Lipids). After growing for 32 hours to OD600 1.1, it was diluted again to OD600 0.1 in 500mL SC +2% Glucose -Adenine + 50µM of t-2-hex and grown for 24 hours to saturation (OD600 2.9). A 500mg cell pellet was suspended in 500µL of Cell Breaking Buffer (consisting of 2% Triton X-100, 1% SDS, 100 mM NaCl, 100 mM Tris-HCl pH 8.0, 1 mM EDTA) and divided into 280µL aliquots. To each aliquot, 200µL of Phenol:Chloroform:Isoamyl alcohol (25:25:1) and 300µL of unwashed glass beads (0.5 mm) were added. Cells were lysed by subjecting them to 10 cycles of vortexing for 30 seconds followed by incubation on ice for 30 seconds. Next, 200µL of TE buffer was added to each lysate, which was then centrifuged at 16100g for 5 minutes at 4˚C. The upper layer (approximately 400µL) was carefully transferred to a new tube, and 2.5 volumes of 100% ethanol were added, followed by mixing by inversion. The DNA was pelleted by centrifugation at 16100g for 5 minutes at 20˚C. The supernatant was discarded, and the DNA pellets were resuspended in 200µL of RNAse A solution (250µg/ml) and incubated for 15 minutes at 55˚C with gentle agitation using a thermoblock at 1000rpm. Then, 2.5 volumes of 100% ethanol and 0.1 volume of 3 M NaOAc pH 5.2 were added, and the samples were mixed by inversion. The DNA was pelleted by centrifugation at 16100g for 5 minutes at 20˚C. The pellets were washed with 70% ethanol under the same conditions, the supernatant was completely removed, and the pellets were dried for 10 minutes at 37˚C. Finally, the pellets were resuspended in a total volume of 100 ml of water and incubated for 10 minutes at 55˚C with gentle agitation using a thermoblock at 700rpm. The DNA was then analyzed on a 0.6% 1X TBE agarose gel using a standard 1 kb GeneRuler, and quantification was performed using Fiji software. 2μg of both genomic DNA were digested using 50U of MboI (DpnII) (Thermo Fisher Scientific) and Hin1II (NlaIII) (Thermo Fisher Scientific) enzymes at 37°C for 24h, in separated reactions. Subsequently, the enzymatic reaction was heat-inactivated at 65°C for 20 minutes. The resulting DNA fragments were circularized by adding 25 Weiss U of T4 ligase (Thermo Fisher Scientific) and ligated at 22°C for 6 hours in a total volume of 400µL. To precipitate the circularized DNA molecules, a mixture of 0.3M sodium acetate (pH 5.2), 1mL of ethanol, and 5μg of linear acrylamide (Ambion AM9520) was introduced. The mixture was then incubated at -20°C overnight. After precipitation, the DNA was pelleted at 16100g and 4°C for 20 minutes, followed by washing with 1mL of 70% ethanol. The DNA was then dried at 37°C for 10 minutes. Each prepared circularized DNA sample was resuspended in 100µL of water. For transposon amplification, PCR was performed using the forward primer P5_MiniDs (AATGATACGGCGACCACCGAGATCTACtccgtcccgcaagttaaatatga) and the reverse primer P7_MiniDS (CAAGCAGAAGACGGCATACGAGATNNNNNNNNacgaaaacgaacgggataaatac). Each primer consisted of two parts: the Illumina adaptor (uppercase letters) and a fragment from MiniDs (lowercase letters). The 8-nucleotide index present in P7_MiniDs allows distinction among different libraries. For PCR, circularized DNA was divided into 10 x 100µL reactions containing: 10µL of ligated DNA, 10μL dNTPs mix 2mM each, 10μL 10x DreamTaq Buffer (Thermo Fisher Scientific), 1μL 100uM primer P5, 1uL 100μM primer P7, 2μL DreamTaq DNA Polimerase (5U/μL) (Thermo Fisher Scientific). PCR was performed under the following parameters: 95ºC 5min, 35x [95ºC 30s, 55ºC 30s, 72ºC 3min], 72ºC 10min. Finally, for each condition (Control, t-2-hex) all PCR reactions were pooled into one Hin1II-digested pool and one MboI-digested pool. 100μL from each pool were purified with CleanNGS magnetic beads (CleanNA) according to manufacturer intstructions.