Ion Torrent Proton sequencing; Comparative analysis of transcripts from mia40-4int mutant and wild-type Saccharomyces cerevisiae strains quantified in RNA-seq experiment
Comparative analysis of transcripts from mia40-4int mutant and wild-type Saccharomyces cerevisiae strains quantified in RNA-seq experiment
Yeast cells (3 X10 power of 8) were isolated by centrifugation at 4500 x g, 19 degree C for 5 min. Cell pellets were resuspended with RNAlater solution (Ambion) and stored at -80 degree C before RNA extraction. Yeast cells were grown in the liquid culture at 19 degree C in the YPG media (3% [v/v] glycerol) till the mid-logarithmic phase (OD600 of approx 0.65) RNA was extracted from frozen material using MagNA Lyser with MagNA Pure Compact RNA Isolation Kit (Roche), according to manufacturer's protocol. Genomic DNA remaining after isolation has been digested with TURBO DNA-freeKit (Life Technologies) according to manufacturer's protocol for standard digestion. To ensure that no gDNA contamination was present, each RNA sample has been used as a template for PCR reaction with primers specific to genomic loci (ERV1 gene). Samples were run on Agilent 2100 Bioanalyzer to ensure that high-quality RNA was used. Accurate RNA concentrations were measured with Qubit RNA BR Assay (Life Technologies). 1.5 ug of RNA from each sample was spiked with 3 ul of 100x diluted ERCC RNA Spike-In Mix (Life Technologies) and rRNA depleted with Ribo-Zero Gold rRNA Removal Kit (Yeast) (Illumina) according to manufacturer's protocol. RNA was validated on Agilent 2100 Bioanalyzer and Qubit and the depletion levels (95%-96%) were estimated . RNA-seq libraries were then prepared with Ion Total RNA-Seq Kit v2 (Life technologies) according to manufacturer's manual with slight modifications. Briefly, RNA was fragmented with RNase III, purified with magnetic beads and validated on Agilent 2100 Bioanalyzer. Fragmented RNA was hybridized and ligated with adapters and reverse transcribed. cDNA was purified with the same magnetic beads and amplified (2 + 10 cycles instead of 2 + 16 cycles in the manual) using barcoded primers. Amplified cDNA was purified and molar concentrations were measured with Agilent 2100 Bioanalyzer. Equal molar concentrations of each sample were pooled to prepare library mix for sequencing.
Experimental Factor: genotype
mia40::mia40-4
LIBRARY_STRAND
second strand