yLIC136_2_dnrp1-2-NlaIII-N715-b
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 HiSeq platform (yLIC136), PCR amplification of the transposon genome-junctions and library preparations were performed by Agnès Michel and Benoît Kornmann (Oxford) according to their established protocols [1].  [1]: Michel AH, Hatakeyama R, Kimmig P, Arter M, Peter M, Matos J, et al. Functional mapping of yeast genomes by saturated transposition. eLife. 2017;6. doi:10.7554/eLife.23570.
ENA first public
2024-11-04
INSDC center name
TU Delft
INSDC status
public
Submitter Id
E-MTAB-14476_2:yLIC136_2_dnrp1-2-NlaIII-N715-b
broker name
ArrayExpress
collection date
not collected
common name
brewer's yeast
genotype
nrp1Δ
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
whole organism
sample name
E-MTAB-14476_2:yLIC136_2_dnrp1-2-NlaIII-N715-b
scientific_name
Saccharomyces cerevisiae
strain
W303