GSM4121384: Rpb3-FLAG rtt109D haploid MATa mRNA seq SPO 4h replicate 2; Saccharomyces cerevisiae; RNA-Seq
Cell pellets were grinded in fine powder using a cryo-mill instrument. Powder was re-suspended in lysis buffer and lysate subected to DNAse I treatment for 20min. Lysate was then clarifed through two rounds of centrifugation. Anti-Flag immunoprecipitation was performed using anti-FLAG M2 agarose beads for 2.5h at 4C on a rotating wheel. Beads were then washed 5 times using cold wash buffer for 5min at 4C. Beads were then cleaned using a Pierce column and elution performed using FLAG peptide (450ul 10mg/ml Sigma FLAG peptide) for 30min at 4C. Elution was then subjected to a phenol:Chloroform extraction and RNA precipitated in ethanol containing 0.3M Na acetate. RNAs were then resuspended in Nuclease free water, quantified by Qubit and analysed by Bioanalyser. At least 500 ng of purified RNA was treated with DNAse and purified on column (Macherey-Nagel). At least 100 ng of purified total RNA was used as the inputs for the KAPA RNA hyperPrep kit (KK8540, Roche). Libraries were prepared according to manufacturer's instructions. After bead based clean up, libraries were sequenced on an Illumina HiSeq 4000 to an equivalent of 75 bases single-end reads, at a depth of approximately 20 million reads per library.
GEO Accession
GSM4121384