GSM1508100: RNA seq of spore pool A; Saccharomyces cerevisiae; RNA-Seq
DNA: see Wilkening et al. 2013 BMC bioinformatics. RNA: see Wilkening et al. 2013 NAR. RNA: Total RNA was isolated by a standard hot phenol method and treated with RNase-free DNaseI using Turbo DNA-free kit (Ambion). For strand specific total RNA-Seq the libraries were prepared as described in Wilkening et al. 2013 NAR which is a modified protocol of Parkhomchuk et al. 2009 NAR. Briefly, 10 µg of total RNA were fragmented by incubating the samples at 80°C for 5 min in the resence of RNA fragmentation buffer (40 mM Tris-acetate, pH 8.1, 100 mM KOAc and 30 mM MgOAc). The fragmented RNA was purified using 1.5× Ampure XP Beads (Beckman Coulter Genomics). Eluted RNA was reverse transcribed with 3 µl oligo dT18 with a VN anchor (1 µM, Invitrogen), 3 µl random hexamers 30 ng/µl, Invitrogen) and 2 µl 10 mM dNTPs. The samples were incubated at 65°C for 5 min and ransferred to ice. 8 µl 5X First strand buffer (Invitrogen), 4 µl DTT 0.1 M, 0.5 µl Actinomycin D (1.25 mg/ml) and 0.5 µl RNasin plus RNase inhibitor (Promega) were added to each sample and the samples were then incubated at 25°C for 2 min. Following this, 0.5 µl Superscript III reverse transcriptase (200 U/µl, Invitrogen) was added. The retrotranscription was carried out at 25°C for 10 min, at 42°C for 50 min,  and inactivated at 70°C for 15 min. After cleanup, the 2nd strand cDNA synthesis was done with dUTPs  instead of dTTPs. For ligation, 1 µl of forked PE multiplexed adaptors (40 µM) was used. The dUTPs of the second strand were hydrolyzed by incubating the samples at 37°C for 30 min with 5 units of UDG in UDG reaction buffer (NEB). The samples were purified using 1X Ampure XP beads. After 10 cycles of PCR amplification and clean up, samples were submitted to the EMBL core facility for 100bp  paired end sequencing on a HiSeq 2000 (Illumina). DNA: A modified version of the PrepEase Genomic DNA Isolation Kit   (Affymetrix, 78855 1 KT) based on enzymatic cell wall digestion was  used for the DNA isolation from yeast cells. All of the mixing steps  were performed by pipetting using a Biomek FX pipetting robot (Beckman  Coulter) in 96-well plates. Cell pellets from 4 deep-well plates, each  containing 1 ml overnight culture, were combined for the DNA  isolation. The Spheroplast and Enzyme Solution from the   kit was replaced by Qiagen   s Y1 lysis buffer (1 M sorbitol, 100 mM  EDTA, pH 8.0, 14 mM   -mercaptoethanol) freshly supplemented with 2.5  l/ml of Zymolyase (Seikagaku Inc.) and 2.5   l/ml RNase A (10 mg/ml,  Qiagen). 200   l of this buffer was added to each pellet, mixed and  incubated at 37  C for 90 min with gentle shaking every 30 min. 200  l of water was added to each well and the plate was centrifuged at  6000 x g for 4 min (for centrifuges with maximum 3,000 x g,  centrifugation times can be tripled) and the supernatant was decanted.  120   l Homogenization Buffer was added and mixed to resuspend the pellet completely. 100   l of chloroform and 400   l of Protein  Precipitation Buffer were added to the lysate and mixed. Plates were  centrifuged at 6,000    g for 15 min. 450   l of the upper aqueous  phase was transferred with the robot (pipetting height was optimized  in advance) to a 1 ml deep-well plate containing 340   l of  isopropanol per well. The solution was mixed, left for 15 min at room  temperature, and centrifuged at 6,000    g for 15 min. After decanting   the supernatant, 1 ml of cold 70% ethanol was added to the pellet,  mixed and centrifuged at 6,000    g for 10 min. The supernatant was decanted, and the tube was placed upside down on a paper towel and dried for 5 min at 37  C. The DNA pellet was resuspended in 300   l of DNA Resuspension Buffer or Elution Buffer (EB, 10 mM Tris HCl) by shaking plates for 30 min at 37  C and later by mixing. DNA fragmentation was done with a Bandelin and a Covaris sonicator,  except for spore pool B, where only a Covaris fragmentation was applied. Instead of the standard column or bead-based cleanup steps,  we heat-inactivated the enzymes used for end repair, dA-tailing, and  ligation, then added the respective enzyme (+ buffer). DNA  Quantification: For quantification of genomic DNA and pre-PCR  libraries in 96-well plates, we used Quant-iT PicoGreen dsDNA Reagent  (Invitrogen) in optical plates (Greiner). The fluorescence was  measured at 485 nm excitation and 535 nm emission in a Genios  microplate reader (Tecan) according to the manufacturers  instructions. The pooled libraries were quantified before and after PCR with a Qubit spectro-fluorometer (Invitrogen) according to the manufacturers instructions. All pre- and post-PCR libraries were run on a High Sensitivity Bioanalyzer chip (Agilent) to determine  the size distribution. The samples were then diluted to 10 nM and  clustered on the Illumina cBot clustering station for paired-end   sequencing on an Illumina HiSeq 2000.
GEO Accession
GSM1508100