7 protocols
All zero values were converted to 0.1. All values were then log2 transformed
Reads were mapped to the P.pastoris genome sequence (October 2010 version) using tophat. Reads mapping to the sense strand were counted using HTSeq in conjunction with the P.pastoris .gtf annotation file (October 2010 annotation). Count data were then normalised in R using the DESeq package.
Output .csfasta and .qual files were generated from within the SOLiD sequencing platform (version 4).
Heterologous protein synthesis was induced in the strains by changing the carbon source composition of the medium to sorbitol (11.3 g/L) plus methanol (9 g/L). Samples were taken immediately prior to changing to the methanol media, 1 hr and 3 hr after methanol induction and finally after reaching a second steady state in the new media.
PolyA-enriched RNA was prepared from total RNA using the Ambion MicroPoly(A)PuristTM Kit (AM1919). Total RNA Sequencing libraries were prepared from polyA-enriched RNA samples using the SOLID RNA barcoding (ABI PN4427046) and Total RNA-seq (ABI PN4445374) kits according to the manufacturers’ recommendations.
Derivatives of Pichia pastoris strain GS115 were grown to steady state in chemostat cultures (1L volume in 2L fermenter; 30oC; pH5; 750 rpm stirring; air flow 1L/min) in a defined carbon-limited medium based on sorbitol (20 g/L). Dilution rates at steady state were 0.004-0.015 L/h, and all other non-carbon nutritional requirements were in excess at constant residual concentrations.
Cells were lysed mechanically at 4oC in the presence of trizol. After partitioning with chloroform, RNA was precipitated from the aqueous phase using isopropanol. RNA was washed with 70% ethanol, and redissolved in water. 10 microg aliquots of the final RNA solution were ethanol precipitated.