2024-08-30
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA24569668
External Id
Department of Cell Biology, Faculty of Science, Charles University
INSDC center name
2016-12-09T09:49:18Z
INSDC last update
public
INSDC status
ERS1470874
SRA accession
E-MTAB-5336:rad6
Submitter Id
ArrayExpress
broker name
baker's yeast
common name
Protocols: Thirty milliliters of yeast cultures were grown at 30 °C in the YPAD medium (1% yeast extract, 2% peptone, 0.01% adenine, 2% glucose). Cells (8 ml of culture) were harvested by centrifugation (1000 g, 3 min, room temperature) at the exponentional growth phase (OD600 ~1.8). Cell pellets were stored at -80 °C. Thaw cell pellets on ice (~5 min). Add 400 ul of extraction buffer (1 mM EDTA, 0.1 M LiCl, 0.1 M Tris-HCl pH 7.5, 1% SDS), 400 ul of glass beads and 400 ul of acidic phenol (pH 4.3):chloroform:isoamylalcohol (25:24:1) solution. Disrupt the cells in FastPrep-24 (MP Biomedicals) (speed 6, duration 25 s) using 3 shaking cycles with 5 minutes incubations on ice in between. Pellet the debris by centrifugation (20 000 g, 10 min, 4 °C). Transfer the supernatant to a new microcentrifuge tube. Add 30 ul of 40% KAc (pH 5.5) and 400 ul of phenol (pH 4.3):chloroform:isoamylalcohol (25:24:1) solution. Mix vigorously by vortexing for 30 s and repeat the centrifugation step. Transfer the upper phase to a new microcentrifuge tube, add 1 ml of 96% EtOH, mix by inverting the tube and let RNA precipitate for at least 1 h at -20 °C. Pellet the RNA by centrifugation (20 000 g, 15 min, 4 °C). Remove the supernatant and rinse the pellet with 70% EtOH. Let the RNA pellet dry. Dissolve the RNA pellet in 150 ul of DEPC-treated water and measure RNA concentration on Nanodrop 2000 (Thermo Scientific). Take the volume corresponding to 150 ug of RNA. Add DEPC-treated water to the final volume of 175 ul, add 20 ul of DNase buffer and 5 ul of DNase from the MasterPure Yeast RNA purification kit (Epicentre). Finish the RNA preparation with this kit according to the manufacturer’s protocol, starting with step 4 in section B of the protocol. After the last rinse with 70% EtOH (step 12), add 500 ul of 70% EtOH to the RNA pellet and store the samples at -20 °C. Strand-specific paired-end RNA-seq libraries were constructed from poly(A)-enriched RNA samples by BGI Genomics using their standard procedure.
description
deletion
genetic modification
MATalpha his31 leu20 lys20 ura30 rad6::kanMX4
genotype
not applicable
isolate
Saccharomyces cerevisiae
organism
Saccharomyces cerevisiae
organism
RAD6 deletion
phenotype
Saccharomyces cerevisiae
scientific_name
rad6
strain
rad6
title