GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5051709

Query DataSets for GSM5051709

Status

Public on Feb 01, 2021

Title

copperTreated_rep2

Sample type

SRA

Source name

copperTreated

Organism

Saccharomyces cerevisiae

Characteristics

treatment: copper treated
input or rerep: ReRep-Seq

Treatment protocol

We developed a copper-resistant yeast strain by culturing cells in the presence of 6 mM copper for approximately 30 generations. Controls were cultured under normal conditions.

Growth protocol

Yeast strains and plasmids used in this study are listed in Table 1 and were grown according to standard procedures in YPD (yeast extract, peptone, and 2% dextrose) at 30°C for all experiments unless otherwise stated. To accommodate S. cerevisiae’s lack of the thymidine salvage pathway, yeast strains in this paper are all stably transformed with NheI linearized p403–BrdU–Inc HIS3, a reconstituted thymidine salvage pathway cassette, consisting of Herpes simplex virus thymidine kinase (HSV–TK) and human equilibrative nucleoside transporter (hENT1), that enables efficient cellular uptake and incorporation of the thymidine analogue BrdU into DNA . Inserts were confirmed by spot assays for sensitivity to 75 µg/ml FUDR and susceptibility to BrdU uptake and genomic fragmentation via Rerep-seq.

Extracted molecule

genomic DNA

Extraction protocol

Rerep-seq was performed as described in . Briefly, In an 8 strip PCR tube 1 – 5 ug high molecular weight genomic DNA was mixed with 2.5uL 10X Hoechst 33258 (0.1mg/ml) and 2.5uL 10X NEB 4 (50 mM Potassium Acetate, 20 mM Tris-acetate , 10 mM Magnesium Acetate, 1 mM DTT pH 7.9@25°C) to a final volume of 24uL; open tubes placed upright in PCR tube rack, covered with a glass plate (3" x 3" glass plate from VWR Vertical Gel Electrophoresis Systems), exposed to 7.5 minutes of glass plate filtered (UVA only) from Stratalinker followed by addition of 0.5uL UDG (5 units of Uracil-DNA Glycosylase), 0.5uL APE1 (10 units of human apurinic/apyrimidinic endonuclease 1) for 2 hours at 37°C. The digested DNA was repaired with NEB’s FFPE DNA Repair Mix for 30 minutes; mixed with 10X loading dye then run on 0.8% agarose gel for 15 minutes. The fragmented DNA ranging from 0.1Kb to 3Kb was gel extracted with Wizard® SV Gel and PCR Clean-Up System with the purified DNA eluted in 50uL nuclease free water.
Sequencing libraries were constructed using the Illumina Nextera DNA Flex Library Prep Kit (catalog number 20018704) following the manufacturers’ protocol and using 14 cycles of amplification for final library construction. Libraries were constructed using dual index barcodes from the Nextera DNA CD Indexes (catalog number 20018707).

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Data processing

Library strategy: Rerep-seq
Fastq files were aligned to SacCer3 using BWA-mem
Sam files were transformed into bam files and blacklisted using samtools (v1.2, RRID:SCR_002105). Blacklists were the same as previously used for Rerep-seq analysis (Menzel et al., 2020). Bedtools (v2.26.0, RRID:SCR_006646) was used to generate bedGraphs
Bedgraphs were binned and normalized by RPM and mitochondrial reads using a custom R script
Replicates were then averaged
The CUP1 gene is exactly duplicated in the yeast genome, so we replaced the CUP1.1 gene copy with ambiguous nucleotides “N” to force alignment to CUP1.2 rather than allow for random alignment between the two copies.
Genome_build: SacCer3, with cup 1.1 removed
Supplementary_files_format_and_content: bedgraph

Submission date

Jan 31, 2021

Last update date

Feb 03, 2021

Contact name

Joshua C Black

E-mail(s)

blacklabucd@gmail.com

Organization name

University of Colorado

Department

Pharmacology