Illumina HiSeq 4000 paired end sequencing; Integration of transcriptome, proteome and phoshpoproteome data elucidates the genetic control of molecular networks
Integration of transcriptome, proteome and phoshpoproteome data elucidates the genetic control of molecular networks
Of the 115 ml of cultures, 15 ml were collected, centrifuged at 2000 g for 3 min at 4°C, transferred into an Eppendorf and snap frozen in liquid nitrogen for transcriptomic analysis.  All media were prepared in a single batch to limit experimental variability. A subset of 129 strains were picked in random series of 16, pre-cultured in in-house made synthetic dextrose medium (S.D., containing per  liter:  1.7  g  yeast  nitrogen base without amino acids (Chemie Brunschwig), 5 g ammonium sulfate, 2% glucose (w/v), 0.03 g isoleucine, 0.15 g valine, 0.04 g adenine, 0.02 g arginine, 0.02 g histidine, 0.1 g leucine,  0.03  g  lysine,  0.02  g  methionine,  0.05  g  phenylalanine,  0.2  g  threonine,  0.04  g  tryptophan,  0.03  g  tyrosine,  0.02  g  uracil,  0.1  g  glutamic  acid  and  0.1  g  aspartic  acid), and then grown in 115 ml fresh S.D. medium at 30ºC until a maximal optical density at 600 nm (OD600) of 0.8 (+/- 0.1). Media for the cultures of allele replacement strains was prepared the same way as for the whole cross but in a separate batch. Growth parameters were unchanged. Total RNA was isolated from deep frozen aliquots of yeast pellets using the RiboPure™ RNA Purification Kit, yeast (Ambion), which includes a DNase treatment to eliminate contamination. RNA quality was assessed using RNA ScreenTape assay (Agilent).   cDNA libraries were prepared from poly(A) selected RNA applying the Illumina TruSeq protocol for mRNA using a total of 1 μg RNA per sample and 14 PCR cycles.