characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2014-04-05T17:00:37Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T17:22:13Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2439270
characteristics.characteristic: External Id
characteristics.text: EMBL European Bioinformatics Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2014-04-05T17:00:37Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T17:22:13Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS430609
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-2456:WT2C
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: baker's yeast
characteristics.characteristic: common name
characteristics.text: Protocols: Wt yeast (BY4741, which is derived from S288c) and delta-ccr4/pan2 (Beilharz et al., RNA. 2007 Jul;13(7):982-97) were single colony streaked on YPAD-agar plates, and then 2 individual colonies each were inoculated in 2ml YPAD medium and grown o.n. at 30C at 180rpm. OD was measured and 7x107 cells were pelleted, resuspended in 2ml Y1 medium (2ul beta-ME, 150ul zymolase) and incubated for 30min at 30C in a waterbath. After centrifugation, the pellet was lysed in buffer RLT and processed according to the yeast protocol of the Qiagen RNeasy kit. Yield and quality of totalRNA was measured with Nanodrop and confirmed by Agilent RNA Nano chip (RIN 10). A DNase digestion was performed on 20ug totalRNA using Turbo DNase (Life Technologies), followed by an RNeasy column cleanup step and elution into 30ul nuclease free water (Ambion). Poly(A) selection: 2 rounds of polyA selection were performed using the Truseq RNA Sample Preparation kit (Illumina) with several modifications. In more detail: 50ul of DNase digested totalRNA was incubated with 50ul RNA Purification beads (oligo-dT) for 5min at 65C, cooled to 4C, and then incubated for 5min at RT in a DNA Engine tetrade 2 (Biorad) thermocycler. Afterwards, beads were separated with a magnet and washed with 200ul Bead Wash buffer, and then eluted in 50ul elution buffer for 2min at 80C. On RT, 50ul of binding buffer were added, placed on a magnet and washed with 200ul Bead Wash buffer, and again eluted in 50ul elution buffer for 2min at 80C. The supernatant containing the mRNA was transferred into a 1.5ml Low Binding tube (Eppendorf) and quantified with the Qubit RNA assay. Spike-in control: 20ul of spike in in vitro transcripts with a known A42 stretch (Pelechano V. et al., Nature. 2013 May 2;497(7447):127-31) were added. A cleanup step was performed using the RNeasy MinElute Cleanup Kit (Qiagen) according to protocol eluting with 14ul water. G-tailing and fragmentation: The USB Poly(A) Tail-Length Assay kit (Affymetrix) was used with several modifications to add a short G stretch to the 3’end of the mRNA. In detail, 14ul mRNA (with spike-in) were incubated with 4ul of 5xTail buffer mix and 2ul 10x Tail Enzyme mix at 37C for 1h and stopped by addition of 2ul 10x Tail stop solution. After an RNeasy Minelute cleanup step and elution into 18ul of water, chemical fragmentation using the NEBnext Magnesium RNA Fragmentation kit (NEB) was performed in 20ul for 3.5min at 94C. Afterwards, the reaction was immediately put on ice, and 2ul of RNA Fragmentation Stop Solution was added. Another RNeasy Minelute cleanup step was performed, eluting into 15ul of water. cDNA synthesis: 1ul TTTTVN (16.7uM) and 1ul CCCCCCTT (50uM) custom primers (both Sigma) were added to the 15ul fragmented RNA, primed by incubation at 65C for 5min and put on ice. 1st strand cDNA synthesis was performed using the 1st strand mastermix (Illumina Truseq RNA kit) and SuperscriptII enzyme (Life Technologies) by incubating at 25C for 10min, 42C for 50min at 70C for 15min, and cooled to 4C in a MJ-Mini thermocycler (Biorad). 2nd strand synthesis was performed by addition of 25ul 2nd strand master mix and incubation for 1h at 16C. NGS library preparation: An AMPure XP (Beckman Coulter) cleanup step was performed by adding 90ul AMPure XP beads, vortexing and incubating for 15min at RT. After separation on a magnet, the supernatant was removed and the beads were washed twice with 200ul 80% ethanol. After air drying for 10min at RT, cDNA was eluted in 50ul Resuspension buffer and stored at -20C. End repair was performed by adding 10ul Resuspension buffer and 40ul Endrepair mix and incubating at 30C for 30min at 750rpm in a thermomixer (Eppendorf). After another AMPure XP cleanup step using 160ul beads and eluting into 17.5ul Resuspension buffer, the A-overhang addition step was performed. 12.5ul A-tailing mix were added and incubated at 37C for 30min at 750rpm, followed by 5min at 70C and put on ice. Adapter ligation used 2.5ul of a 1:40 dilution of Adapter oligo mix (Agilent Sureselect v4 kit), 2.5ul Resuspension solution and 2.5ul DNA ligase mix, incubated at 30C for 10min in a water bath and stopped by addition of 5ul Stop ligase mix. Adapter ligated cDNA library was cleaned up by 2 rounds of AMPure XP using first 42ul of AMPure XP beads and eluting in 50ul Resuspension buffer and then using 50ul AMPure XP beads and eluting in 40ul Resuspension buffer. Next, a size selection step was performed to create 4 technical replicates between 300-325bp, 325-350bp, 400-425bp and 425-450bp. For this, the purified adapter ligated cDNA was loaded on a 1.5% Agarose gel and separated for 2h at 120V. Using SybrSafe (Invitrogen) as dye and a Dark Reader transilluminator (Clare Chemical Research), narrow bands were cut using 100bp marker as guidance and gel extracted using the Nucleospin Gel and PCR Cleanup kit (Macherey-Nagel) and eluted in 22ul NE buffer. Two sequential rounds of PCR were performed to introduce barcodes and amplify the cDNA library. First, the Sureselect primer and Sureselect ILM Indexing Pre-Capture PCR reverse primer were used with the Phusion HF 2x mix in 50ul in a MJ Mini thermocycler as follows: 2min 98C, followed by 5 cycles of 30sex 98C, 30sec 65C, 1min at 72C, and a final 72C step for 10min. Next, an AMPure XP cleanup step was performed using 90ul AMPure XP beads and eluting into 23ul water. Afterwards, a 2nd PCR was performed using the Sureselect ILM Indexing Post Capture Forward PCR primer and a different PCR Primer Index primer/sample together with the Phusion HF 2x mix in 50ul in a MJ Mini thermocycler as follows: 2min 98C, followed by 13-15 cycles of 30sex 98C, 30sec 57C, 1min at 72C, and a final 72C step for 10min. After a final AMPure XP cleanup step using 90ul beads, the final library was eluted in 20ul water, quantified with the Qubit HS dsDNA kit and pooled equimolarly.
characteristics.characteristic: description
characteristics.text: wild type
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005168
characteristics.characteristic: genotype
characteristics.text: WT2
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001103
characteristics.characteristic: individual
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: organism
characteristics.text: Saccharomyces cerevisiae
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_4932
characteristics.characteristic: organism
characteristics.text: Saccharomyces cerevisiae
characteristics.characteristic: scientific_name
characteristics.text: fresh specimen
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/OBI_0000971
characteristics.characteristic: specimen with known storage state
characteristics.text: BY4741
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0000098
characteristics.characteristic: strain
characteristics.text: WT2C
characteristics.characteristic: title