FD7_4
Saccharomyces cerevisiae
Protocols: ing homologous recombination, followed by counterselection against the URA3 marker with 5-fluoro-orotic acid to obtain a clean gene deletion. bem3∆ and nrp1∆ strains were created using homologous recombination to replace the endogenous genes with the natMX4 and hphMX4 cassettes, respectively. Strains were stored at-80°C as frozen stocks in 40% (v/v) glycerol.  SATAY libraries were generated based on the procedure described by Michel et al. [1], which is a modification of the original SATAY protocol to allow transposition to occur in liquid media. ade− cells were transformed with plasmid pBK549, which was a kind gift from Benoît Kornmann, according to a lithium acetate transformation protocol [2]. To screen for clones transformed with the intact version of plasmid pBK549, 12-24 colonies were picked from the transformation plate, re-streaked on fresh SD-ADE and SD-URA plates and incubated for 3 days at 30°C. For clones that showed full growth on SD-URA plates while producing a small number of colonies on SD-ADE plates, cells were scraped from the SD-URA plate and used to inoculate 25 ml of preculture media (Table S2) at an OD600 of 0.20-0.28. Precultures were grown on an orbital platform shaker at 160 rpm, 30 °C until the OD600 was between 5-7 (∼20h). The saturated precultures were used to inoculate 200 ml of induction media at an OD600 of 0.10-0.27 and grown for 52 hours to allow transposition to occur. The efficiency of transposition was monitored by plating samples of the liquid induction cultures on SD-ADE at T=0 and T=52 hours and scoring the number of colonies on these plates after 3 days of incubation at 30 °C. After 52 hours of induction, the resulting transposon mutagenesis libraries were reseeded in 3 liters of reseed media at an OD600 of 0.21-0.26. Typically, this meant that around 7 million transposon mutants were reseeded per library. Reseeded libraries were grown for 92 hours at 140 rpm, 30 °C. At the end of reseed, cells were harvested by centrifugation of the reseed cultures at 5000 xg for 30 minutes. Cell pellets were stored at-20 °C.  [1]: Michel AH, van Schie S, Mosbach A, Scalliet G, Kornmann B. Exploiting homologous recombination increases SATAY efficiency for loss- and gain-of-function screening. bioRxiv [Preprint]. 2019 [cited 2024 January 9]; p. 866483. doi:10.1101/866483.  [2]: Gietz RD, Schiestl RH. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols. 2007;2(1):31–34. doi:10.1038/nprot.2007.13. A 500 mg frozen cell pellet was resuspended in 500 µl cell breaking buffer (12% Triton X-100, 1% SDS, 100 mM NaCl, 100 mM Tris-HCl pH8.0, 1 mM EDTA) and distributed into 280 µl aliquots. 300 µl of 0.4-0.6 mm glass beads (Sigma-Aldrich, G8772) and 200 µl of Phenol:Chloroform:isoamyl alcohol 25:24:1 (Sigma-Aldrich, P2069) were added to each aliquot and cells were lysed by vortexing the samples with a Vortex Genie 2 at maximum speed at 4°C for 10 minutes. 200 µl of TE buffer was added to each lysate, after which the samples were centrifuged at 16100x g, 4°C for 5 minutes. After centrifugation, the upper layer (∼400 µl) was transferred to a clean eppendorf tube. 2.5 volumes of 100% absolute ethanol was added to each sample and mixed by inversion to precipitate the genomic DNA. After precipitation, the DNA was pelleted by centrifugation at 16100x g, 20°C for 5 minutes. The supernatant was removed and the DNA pellet was resuspended in 200 µl of 250 µg/ml RNAse A solution (qiagen, Cat. No. 19101). the resuspended DNA pellets were incubated at 55°C for 15 minutes to allow digestion of the RNA. After digestion, 20 µl 3M, pH 5.2 sodium acetate (Merck) and 550 µl 100% absolute ethanol was added to each sample and mix by inversion. DNA was pelleted by centrifugation at 16100x g, 20°C for 5 minutes. Pellets were washed with 70% absolute ethanol and dried at 37°C for 10 minutes or until all ethanol had evaporated. The dried pellets were resuspended in a total volume of 100 µl MiliQ water and the concentration of the genomic DNA samples was quantified on a 0.6% agarose gel using the Eurogentec Smartladder 200bp-10kb as a reference. Prepared DNA samples were stored at-20°C or 4°C until used. To prepare genomic DNA samples for sequencing, 2x2 µg of DNA from each sample were transferred to non-stick microcentrifuge tubes and digested with 50 units of DpnII and NlaIII in a total volume of 50 µl for 17 hours at 37°C. After digestion, the restriction enzymes were heat-inactivated by incubating the samples at 65°C for 20 minutes. Digestion results were qualitatively assessed by visualization on a 1% agarose gel stained with Sybr-Safe. Successfully digested DNA samples were circularized in the same tube using 25 Weiss units of T4 DNA ligase (Thermo Scientific, Catalog #EL0011) at 22°C for 6 hours in a total volume of 400 µl. After ligation, the circularized DNA was 693 precipitated using 1ml 100% absolute ethanol, 20 µl 3M, pH 5.2 sodium acetate (Merck) and 5 µg linear acrylamide (invitrogen, AM9520) as a carrier. DNA was precipitated for at least 2 days at-20°C. Precipitated DNA was pelleted by centrifugation for 20 minutes at 16100x g at 4°C and washed with 1 ml of 70% ethanol. After washing, the DNA was re-pelleted by centrifugation for 20 minutes at 16100x g at 20°C, the supernatant was removed and pellets were dried for 10 minutes a 37°C. Each dried pellet was resuspended in water and used as a template for 20 PCR reactions of 50 µl. For samples sequenced on the Illumina NovaSeq 6000 platform (yWT01a, yLIC137), the transposon-genome junctions were amplified using the barcoded primers 1 and 2 [2] for DpnII digested DNA or primers 3 and 4 [2] for NlaIII digested DNA on a thermal cycler (Bio-Rad C1000 Touch). PCR amplified samples were purified using the NucleoSpin Gel and PCR cleanup kit (Macherey-Nagel) and quantified on the NanoDrop 2000 spectrophotometer (Thermo Scientific). For each sample, equal ratios (w/w) of DpnIII and NlaII digested DNA were pooled. Library preparation was performed by Novogene (UK) Company Limited.  Sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit, omitting the size selection and PCR enrichment steps.  [1]:
ENA first public
2024-11-04
INSDC center name
TU Delft
INSDC status
public
Submitter Id
E-MTAB-14476_2:FD7_4
broker name
ArrayExpress
collection date
not collected
common name
brewer's yeast
genotype
wild type genotype
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
whole organism
sample name
E-MTAB-14476_2:FD7_4
scientific_name
Saccharomyces cerevisiae
strain
W303