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Sample GSM7708611

Query DataSets for GSM7708611

Status

Public on Oct 19, 2023

Title

gal80Δ, midlog+16h, fructose, B

Sample type

SRA

Source name

FY4

Organism

Saccharomyces cerevisiae

Characteristics

cell line: FY4
genotype: deleted gal80
treatment: 0.1% fructose
time: mid-log + 16 h

Growth protocol

(1) FY4 (Brachmann et al, Yeast, 1998) cells were pre-cultured in media supplemented with 2% (w/v) sodium pyruvate in a 30C shaking incubator at 180 rpm for 40 hours, and diluted 6-fold with fresh media 6 hours before the experiment. (2) When the experiment began, the cells were washed twice with minimal (Delft) media without carbon sources and then inoculated into 250 mL flasks with 25 mL minimal (Delft) media supplemented with desired concentrations of fructose and palatinose. The volume of inoculated cells was calculated such that the initial OD was 0.05, and then the volume of each culture was topped up to 26 mL. (3) The cultures were then incubated in a 30C shaking incubator at 180 rpm.

Extracted molecule

total RNA

Extraction protocol

For each sample, we harvested x mL of cells, such that the value of OD*x is around 4, by centrifuging the cells at 3500 rpm for 3 minutes at 4C. The supernatant was then removed and the cell pellets were stored in -80C. (2) We followed a column-based protocol on protocol.io to extract RNA: dx.doi.org/10.17504/protocols.io.beetjben, starting from Step 16 without using the deep well plate. Sample quality control was performed on the Fragment Analyser Automated Capillary Electrophoresis System (Agilent Technologies Inc, #5300) with the Standard Sensitivity RNA Analysis Kit (#DNF-471-0500) for quality, and on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) with the Qubit RNA broad range assay kit (#10210) for quantification. The Qubit dsDNA HS assay kit (#Q32854) was used to quantify DNA contamination.
Libraries were generated from 400 ng of each total RNA sample with the QuantSeq 3' mRNA Library Prep Kit REV for Illumina (Lexogen Inc, #016) according to the manufacturer's protocol. A Custom Sequencing Primer (CSP Version 5, included in the kit) was used for Read 1. The libraries were then quantified by fluorometry with the Qubit dsDNA High Sensitivity assay, followed by assessment of quality and fragment size with the Agilent Fragment Analyser with the SS NGS Fragment 1--6000bp kit (#DNF-473-33). Sequencing was performed on the NextSeq 2000 platform (Illumina Inc, #20038897) using NextSeq 1000/2000 P2 Reagents (100 Cycles) v3 (#20046811).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Data processing

The data processing pipeline is written in Nextflow and the repository is available at: https://git.ecdf.ed.ac.uk/s1856140/rnaseq-paired-end
Run FastQC v0.11.9 to produce a quality control report for the input data for every sample
Cut sequencing adapters from 3' end of gene with Cutadapt v1.18
Align reads to genome with HISAT2 v2.1.0
Turn unsorted aligned SAM files into sorted indexed compressed BAM files with SAMtools v1.11
Make bedgraphs showing coverage of aligned reads with BEDTools v2.30.0.
Run featureCounts (in Subread v2.0.0) to count aligned reads to genes for all processed samples
Run multiQC v1.13 to collate single quality control report across all samples.
Assembly: R64-1-1
Supplementary files format and content: CSV file that includes the counts per million reads (CPM) of each gene in each sample in a tidy format.

Submission date

Aug 14, 2023

Last update date

Oct 19, 2023

Contact name

Peter S Swain

E-mail(s)

[email protected]

Organization name

University of Edinburgh

Department

School of Biological Sciences