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Sample GSM1294007

Query DataSets for GSM1294007

Status

Public on Aug 19, 2015

Title

Wild-type GAL (rep2)

Sample type

SRA

Source name

Wild-type GAL

Organism

Saccharomyces cerevisiae

Characteristics

strain backround: BY4741

Treatment protocol

To induce a carbon source shift, BY4741 cells were grown overnight in 100 ml rich glycerol/ethanol media (2% peptone, 1% yeast extract, 3% glycerol and 2% ethanol) to an OD600 of 0.8 at 30 °C with shaking. At the start of the experiment 10 ml of culture was harvested, washed in 1ml of icecold dH2O and snap frozen. To induce GAL gene expression 40% galactose was add the culture to a final concentration of 2%. After 10 minutes, 10 ml of culture was harvested and 40% glucose was added to a final concentration of 2%. Additional 10 ml samples were harvested after 10 and 20 minutes of growth in the presence of glucose.

Growth protocol

The yeast strains BY4741 and Δccr4::KanMX were grown in rich media (2% peptone, 1% yeast extract and 2% glucose) to an OD600 of 0.8 at 30 °C with shaking.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from snap frozen cell pellets by hot-phenol extraction. 1 microgram of this was input into the library preperation.
Library construction was by the PAT-seq approach. Breifly this involves a modified the ePAT approach to tagging adenylated RNA to generate libraries suitable for deep sequencing. Briefly, adenylated RNA is sequence specifically extended by dNTPs using Klenow polymerase and an annealed DNA anchor oligonucleotide. This takes advantage of the native function of DNA polymerase to extend an RNA primer from a DNA template in second strand synthesis. Importantly, any unwanted priming to internal poly(A)-tracts in RNA is avoided by a requirement for 3’ extension in subsequent fragment selection and reverse transcription. No ribosome depletion is necessary. Here, the anchor sequence was compatible with the Illumina index primers and included a 5’ biotin moiety to facilitate handling. In a second step, the 3’ tagged RNA was subject to limited fragmentation by RNase T1. This cleaves RNA after G-residues and ensures that cleavage is only possible within the body of the RNA, not the poly(A)-tract or the DNA sequence of the extended tag. The fragmented RNA was 5’ phosphorylated to allow RNA Ligase 2 mediated ligation of an Illumina compatible splinted-linker to the RNA fragments. Reverse transcription was primed from the anchor sequence. Note: All manipulations after limited fragmentation were performed in association with streptavidin magnetic beads. The cDNA PAT-seq libraries was eluted from beads, size-selected by Urea PAGE and amplified with primers that introduce the features for directional Illumina sequencing and indexing. In samples analysed here, the window of selection was between 120-300 bases. This size range was selected to allow for ≥ 25 bases of 3’UTR sequence to map reads to the genome, the an average yeast poly(A)-tail of ~25 bases (maximum ~90 bases), the majority of reads would contain heterogeneous 5’ sequence of sufficient length to map uniquely to the yeast genome. Note: all reads run in 5’ to 3’ direction from unique sequence into a variable length of poly(A) homopolymers. This means that color balance is preserved and that any low fidelity within the homopolymers is limited to the end of the read.
Cluster Generation: 9pM of libraries per lane using Illumina c-bot. Illumina protocol 15006165 Rev J, July 2012
Sequencing chemistry: 1 x 100bp sequencing using Illumina protocol 15035788 Rev A, Oct 2012

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 1500

Description

Illumina index 9
GALRep2

Data processing

Library strategy: PAT-Seq
All analysis was carried out using Tail Tools version 0.17, available from https://pypi.python.org/pypi/tail-tools/0.17.
Reads were clipped of poly(A) tail and end adaptor sequence.
Reads were aligned using SHRiMP 2.2.3. Where a read had several equal best alignments, one alignment was chosen at random.
Alignments were extended where there truly was a poly(A) sequence in the reference.
Alignments were assigned to genes. The alignment was allowed to be up to 200 bases downstrand of the annotated transcription end point and still be counted as belonging to that gene. From this counts of the number of reads aligning to each gene were produced, and the average poly(A) tail length observed in reads having a poly(A) tail of length at least 4 (not counting what was present in the reference sequence) was calculated.
Genome_build: sacCer3
Supplementary_files_format_and_content: count.csv: Count matrix - read count aligning to each gene.
Supplementary_files_format_and_content: tail.csv: Tail length matrix - average poly(A) tail length observed in reads having a poly(A) tail of length at least 4. At least 10 reads were required for an average to be given. Note that this is probably an underestimate of the poly(A) tail length, as the poly(A) sequence may extend beyond the length of the read.

Submission date

Dec 18, 2013

Last update date

May 15, 2019

Contact name

Traude Beilharz

E-mail(s)

traude.beilharz@monash.edu

Organization name

Monash University

Department

Biomedicine Discovery Institute