2024-09-30
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114856399
External Id
Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Barcelona, Spain
INSDC center name
2023-12-07T14:37:30Z
INSDC last update
public
INSDC status
ERS17281266
SRA accession
E-MTAB-13613:Strain 3
Submitter Id
ArrayExpress
broker name
not collected
collection date
baker's yeast
common name
Protocols: The Saccharomyces cerevisiae PPY1 strain (MATa his3∆0 leu2∆0 met15∆0 ura3∆0 bar1::leu2) was transformed with the appropriate DNA fragments to generate all the mutant strains used in this work. The PPY1 strain was obtained from Oscar Aparicio’s lab at the University of Southern California, USA. For the selection of the mutant strains, we used YPD with or without 5-FOA (5-Fluoroorotic acid) and SD (Synthetic Defined) with the required amino acids. We generated 4 mutant strains, with the 81-nt DNA sequence (5'-GCGTGTTGTGTTTTCTCCGAGGAGAAACATTCAAATCTTGTGCTATGGCTTTGCCTACCGTCTGCGCCATCCATCTTTCGC-3') inserted in the coding sequence of 2 selected genes per strain (see Suppl. Table S1). We selected four non-essential genes which showed phased nucleosomes (UBX5, CKB2, PPT1, TRP4; see definition below) together with four non-essential control genes which were not-phased (BSP1, DGK1, SLM3, PAN5). In order to determine the correct incubation time to inhibit transcription without killing the cells, we selected 2 genes with low RNA stability (RPA135 and NMD3) and 2 genes with high RNA stability (ACT1 and DGK1) to serve as controls34. We then measured their mRNA level by qPCR after incubation with 10-phenanthroline at 100 µg/ml at 30ºC during 0, 5, 15, 30 and 45 minutes. Using this approach, we observed that the amount of RPA135 and NMD3 mRNA started to decrease after 30 min. This incubation time was selected to perform the MNase-seq experiments on cells with inhibited transcription. Cell lysis followed by Phenol-Chloroform extraction The sample preparation was done using the Illumina TruSeq DNA sample preparation kit for whole genome sequencing, following the Illumina standard protocol.
description
MATa his3∆0 leu2∆0 met15∆0 ura3∆0 bar1::leu2
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Saccharomyces cerevisiae
organism
Saccharomyces cerevisiae
organism
Saccharomyces cerevisiae
scientific_name
PPY1 strain with 81bp in PPT1 and SLM3
strain
Strain 3
title