GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2462584

Query DataSets for GSM2462584

Status

Public on Apr 11, 2017

Title

Rpo21 glucose to no glucose anchor away strain rapamycin time point 14

Sample type

SRA

Source name

Rpo21 glucose to no glucose anchor away strain rapamycin time point 14

Organism

Saccharomyces cerevisiae

Characteristics

genotype: HHY168: MATalpha; tor1-1; fpr1::NAT PMA1-2xFKBP12::TRP
molecule subtype: UV cross-linked RNA

Growth protocol

For an 8 time-points time-course, 8L of cells were grown in synthetic medium with glucose (SD-TRP) to exponential phase (OD600 ~0.5) at 30ºC. For time-point zero, 1L of cells were cross-linked in the Vari-X-linker using the high-output 254nm lamps for 12 seconds and then harvested by rapidly passing the cells through a 0.8 µm filter (Millipore) using a home-made vacuum filtration device (contact UVO3 for information and prices). The remaining 7L of cells were harvested on filters and quickly resuspended in S-TRP (no glucose samples) or SD-TRP (glucose control samples) and maintained at 30oC. For each time-point 1L of cells were cross-linked in the Vari-X-linker and harvested by filtration as above. This yielded ~1g of cells for each time-point.

Extracted molecule

total RNA

Extraction protocol

Cells were lysed in 1V/w of TN150 (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 5 mM b-mercaptoethanol) and 3V of Zirkonia beads (0.5 mm; Thistle Scientific) as previously described (Granneman et al. PNAS 2009). For the Nab3 anchor-away nuclear depletion experiments (Haruki et al., Mol Cell 2008), cells were incubated with 1µg/ml rapamycin (Sigma) for 1 hour before the cells were shifted. For the Pol II (Rpo21-HTP) cCRAC experiments, cells were lysed in 1V/w TMn150 (50 mM Tris-HCl pH 8.0, 10 mM MnCl2, 0.1% NP-40, 5 mM b-mercaptoethanol, 150 mM NaCl, Roche Midi protease inhibitors; 1 ml per gram of cells). Subsequently, 1V/w of TMn150 was added containing 1U/ml of RQ1 RNase-free DNAse (Promega) and the suspension was incubated for half an hour on ice to degrade the chromatin. Three milliliter of lysis buffer was added and extracts were clarified by centrifugation (20 min at 4,500 g and 20 min at 20,000 g at 4 °C). Extracts were incubated with 250 µl of equilibrated IgG Sepharose beads (GE Healthcare) for 2h at 4 °C.
linker ligations were performed as previously described (Granneman et al., PNAS 2009) using adapters provided in the Supplementary Information of the manuscript. Cross-linked protein-RNA complexes were resolved on 1mm thick 4-12% NuPAGE gels (Thermo Fisher Scientific), transferred to nitrocellulose membranes and visualized by autoradiography. Bands corresponding to the size of the protein of interest, including a region 0.5 cm above the band, were cut from the nitrocellulose membrane and pooled in a single 2 ml tube. Radiolabeled RNA was extracted by incubating the membrane slices with 200 µg of proteinase K in 800 µl of wash buffer II containing 1% SDS and 5 mM EDTA. The solution was transferred to a new tube and the RNA was subsequently phenol-chloroform-extracted and ethanol-precipitated. Reverse transcription with SuperScript III was performed as per manufacturer's procedures (Thermo Fisher Scientific) using the reverse transcription primer listed in Table S2. The cDNAs were purified using the Zymo DNA Clean & Concentrator 5 kit and eluted into a final volume of 10 µl. Five µl of cDNA was PCR amplified using Pfu polymerase (Promega) for 20-24 cycles (95ºC 30s, 52ºC 30s and 72ºC 1min) using PCR oligonucleotides listed in Table S2. PCR products were resolved on 2% Metaphor agarose gels (Lonza) and 160-300 bp fragments were gel purified using the miniElute kit (Qiagen) according to manufacturer’s procedures. Paired-end sequencing (50 bp) was performed by Edinburgh Genomics using the Illumina HiSeq 2500 and 4000 platforms.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina MiSeq

Description

CRAC library
Rpo21_glu_to_noglu_14_RAP
processed data file:
Rpo21_RAP_no_RAP_0_14_control_exp_data.txt

Data processing

CRAC data processing steps: All processing steps were performed using the pyCRAC package version 1.2.2.6. Demultiplexing was performed using pyBarcodeFilter.py (version 2.3.4). 3' adapter removal and 3' end quality trimming (min Phred = 30) was performed using FlexBar (version 2.5). Reads were collapsed using random nucleotides present in 5' barcodes using pyFastqDuplicateRemover.py (version 0.0.2). Reads were mapped to the S. cerevisiae geneome (EF.64; source ENSEMBL) using novoalign (version 2.0.4) and feature counts were generated using pyReadCounters (version 0.5.3).
We scaled the FPKM values of all transcripts within each time point by a constant factor such that the sum of FPKMs for all time points and all experimental replicates is the same. This was done for all data sets that were analysed simultaneously, e.g. Nab3 and Pol II data sets (see below). For all data sets, the same time points were used (on a few occasions, temporally close time points were deemed identical for experimental purposes). Finally, for all analysed data sets, we divided each time series of each experimental replicate by its steady state value before the imposition of stress (at 0 minutes after the nutrient shift). This way, all time series start at the same normalised binding value of 1 a.u. before the nutrient shift and the other values for later time points are relative to the background binding signal. We only keep those transcripts for the analysis that have real values for all time points in all experimental replicates after all steps of the normalisation procedure.
The scaled FPKM values are provided in the processed data files.
Genome gtf and fasta files are also included
Genome_build: EF64
Supplementary_files_format_and_content: text files containing normalized FPKM values for each time-series

Submission date

Jan 18, 2017

Last update date

May 15, 2019

Contact name

Sander Granneman

E-mail(s)

[email protected]

Organization name

University of Edinburgh

Department

Centre for Synthetic and Systems Biology