RNA_Seq of S. graminicola: fermentation 72H non Fatty acid_Rep2
After 48 hours of growth at 30C and 120 rpm, approximately 50 ml of S. graminicola seed culture was inoculated into two 5 L fermenters (Applikon) containing 2 L of producing media (20g/L of glucose, 3g/L of NaNO3, 0.3 g/L of KH2PO4, 0.3 g/L of MgSO47H2O and 1 g/L of yeast extract). The only difference between the two fermenters is that one also included fatty acid (FA) (80g/L) for the induction of MEL production. During incubation one fermenter was feed with glucose (non-induced) and one with both FA and glucose (induced). The sugar feeds were at the rate of 2g/hr/3L using a 50% glucose solution filter sterilised. The FA feed was at 2g/hr/3L. The fermentation was run for 117 hours and samples of 50 ml were taken from each fermenter at 24, 48, 72, 96 and 117 hours. The fermentations were conducted twice, independently, but due to the failure of an air compressor in one of the fermenters the first experiment stopped after 72 hours. This means we got, for each condition, duplicate samples for 24, 48, 72 hours and a single sample for 96 and 117 hours. We then harvested the cells by centrifugation, washed twice with PBS (to remove excess of FA from the media) and immediately frozen with liquid nitrogen. RNA extraction utilised the RNeasy Midi Kit (Qiagen Hilden, Germany), according to the manufactures protocol. The frozen pellet was ground in a mortar and pestle with liquid nitrogen. The powder was resuspended in 4 ml of lysis buffer RLT which was supplemented with 0.01%v/v of -mercaptoethanol. As genomic DNA depletion is already part of this kit no extra DNAse treatment was perform. The concentration of the RNA was measure using the Qubit HS RNA assay (Thermo Scientific) and the integrity was analysed with the Agilent 2100 Bioanalyzer using the RNA pico kit (Agilent, Waldbronn, Germany). For the RNA depletion of total RNA we used the NEBNext Poly(A) mRNA magnetic isolation module (New England BioLabs, Ipswich, MA, USA) according to the manufacturers protocol. We used 5 g of total RNA. The concentration of the mRNA was measure using the Qubit HS RNA assay (Thermo Scientific) and the integrity was analysed with the Agilent 2100 Bioanalyzer using the RNA pico kit (Agilent, Waldbronn, Germany). Only samples that reached a ribosomal integrity number (RIN) higher than 8 were used for the next step. The NEBNext Ultra Directional kit (New England BioLabs, Ipswich, MA, USA) was used for the cDNA synthesis with 1 to 0.6 ng of mRNA as input material. A total of 17 cycles of PCR amplification was conducted using multiplex primers (indexed primer used to give a unique identifier to each library). The concentration of the cDNA libraries was measure using the Qubit HS DNA assay (Thermo Scientific) and the integrity was analysed with the Agilent 2100 Bioanalyzer using the DNA kit (Agilent, Waldbronn, Germany). All the libraries were pooled to be approximately equimolar. The RNAseq libraries were pooled into one lane and run as paired-end 100 bp reads on the HiSeq4000 platform using the SBS v4 chemistry. These aspects of the work were all conducted by the CGR (Liverpool).
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