GSM4144959: rad6E49K-3V5 repeat 3; Saccharomyces cerevisiae; RNA-Seq
Total RNA was isolated using the acid-phenol method. Yeast cells were grown to OD600nm 0.7 and harvested (10.5 X 107), washed with RNase-free water and stored at -80°C. Frozen cells were resuspended in 0.3 ml TES buffer (10 mM Tris.Cl pH 7.5, 10 mM EDTA, 0.5% SDS), combined with equal volume of Phenol pH 4.3 (Thermo Scientific) and incubated at 65°C for 1h with agitation (140 rpm) in a Thermomixer (Eppendorf). The lysate was cooled on ice for 10 min and centrifuged (13200 rpm, 10 min at 4°C). Supernatant (0.25 ml) was combined with equal volume of 25:24:1 Phenol-choloroform-isoamyl alcohol pH 4.3 (Thermo Scientific), mixed well by vortexing and end-over-end rotation for 10 min at 4°C, and centrifuged (13200 rpm, 10 min at 4°C). Supernatant (0.2 ml) was overlaid onto 0.8 ml 5.7M CsCl (Sigma) and centrifuged in a Beckman Optima Tabletop Ultracentrifuge with MLA-130 rotor (30000 rpm, 18h at 4°C). The pellet was dissolved in 0.2 ml RNase-free TE pH 7.0 (Ambion) by incubating for 1h at 4°C, before precipitation using ethanol and sodium acetate pH 4.3 (Ambion) and centrifuged (13200 rpm, 20 min at 4°C). The pellet was washed with 70% ethanol, dried and dissolved in 0.1X TE. Equal amount of RNA (50 μg) was further purified using Qiagen RNeasy Mini Kit by following the manufacturer's recommendations. Purified RNA was used in library preparation with Illumina TruSeq HT Stranded mRNA Library Preparation Kit with PolyA Selection. For RNA isolated from strains DHY217 and YMC203, Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold rRNA Removal Kit Yeast was used. The libraries were subjected to 50 bp single-read sequencing in HiSeq 2500. Three independent RNA isolates from each yeast strain were sequenced. Intact poly(A) RNA was purified from total RNA samples (100-500 ng) with oligo(dT) magnetic beads and stranded mRNA sequencing libraries with dual index adapters were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation Kit (RS-122-2103).  Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (cat# 5067-5582 and 5067-5583).  The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant Kit (cat#KK4824).  Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis. RNA-seq: llumina TruSeq HT stranded mRNA library preparation with Poly A selection to facilitate the use of dual index. Illumina TruSeq Stranded total RNA with Ribo-Zero Yeast protocol was followed for library preparation
GEO Accession
GSM4144959