GSM1634053: EV strain 1; Saccharomyces cerevisiae; Bisulfite-Seq
5 OD600 of yeast cells were disrupted by vortexing for 6 minutes in a Disruptor Genie (Scientific Industries, Inc.) in the presence of an equal volume of breaking buffer, acid-washed glass beads and phenol:chloroform (1:1). After the addition of TE buffer, aqueous phase is transferred into a new tube and precipitated with ethanol. The nucleic acid pellet is resuspended in TE buffer and treated for 1 hour at 37°C with RNase A, followed by incubation for 1 hour with 2 mg/ml Proteinase K in the presence of 1% SDS at 60°C. The resulting solution is treated twice with phenol:chloroform (1:1), once with chloroform and ethanol precipitated. The DNA pellet is resuspended in EB buffer (Qiagen). Between 500-1000 ng of extracted yeast DNA are added to 2 ng of λ unmethylated DNA (Promega, D1521) and the mixture is sonicated with a Covaris S-2 to obtain fragments in the 200-300 bp range [Total Time: 6 minutes; Duty Cycle: 10%; Intensity: 5; Cycles/Burst: 200; Mode: Frequency Sweeping]. The reagents used in the library preparation are from the Illumina TruSeq DNA Sample Prep kit v2. End-Repair, purification and dA-tailing steps are performed according to manufacturer’s instructions. Ligation is performed according to the protocol except that 1 μl of Illumina TruSeq Adapters is used in the final reaction. The ligation reaction is purified using 1.2 volumes of AMPure XP beads (Beckman Coulter Inc.) and DNA fragments with a 170-350 bp range are enriched using 0.7 and 0.3 volumes of AMPure XP beads in the first and second size-selection step, respectively. Samples are treated with bisulfite (EpiTect kit, QIAGEN) according to manufacturer’s protocol, except that two consecutive rounds of conversion are performed, for a total of 10 h of incubation. Half of the converted DNA is amplified using MyTaq Mix (Bioline) and Illumina TruSeq PCR Primer Cocktail according to the following protocol: initial denaturation at 98°C for 30 seconds; 12 cycles of 98°C for 15 seconds, 60°C for 30 seconds, 72°C for 30 seconds; final extension at 72°C for 5 minutes. The final product is purified using AMPure XP beads before being submitted for sequencing.
GEO Accession
GSM1634053