Illumina HiSeq 4000 paired end sequencing; Transcriptional and physiological diurnal responses of Tetradesmus obliquus under light-dark cycles and nitrogen limitation
Transcriptional and physiological diurnal responses of Tetradesmus obliquus under light-dark cycles and nitrogen limitation
After steady state was reached, 8mL samples were taken for RNA extraction (approximately 10 mgDW). Cells were immediately collected by centrifugation (4255 xg, 0°C for 5 min), supernatant was discarded and pellets were frozen in liquid nitrogen and stored at -80°C until further extraction. Samples for RNA extraction were taken in intervals of 1h for WT or every 3h for slm1. 16:8h diurnal cycle adapted cells. Dilution medium (Breuer et al., 2013) was prepared without KNO3 and KNO3 was separately fed at 0.075 gN·L-1day-1 for the wild-type and 0.052 gN·L-1day-1 for the slm1. This corresponds to a nitrogen limitation of 30% of the nitrogen consumption rate observed under nitrogen replete and light limitation (Remmers et al., 2017). Dilution medium was only added during the light period and switched off during the night. RNA extraction was performed using the Maxwell® 16 LEV simplyRNA Tissue kit (Promega). Frozen algae pellet (≈200 μL) were submerged in 400 μL of homogenizing buffer supplemented with 8 μL 1-thioglycerol in a 2 mL Lysing matrix C tube (MP), prefilled with a mix of glass beads. Samples were disrupted using a FastPrep-24 instrument (MP). After disruption, all liquid was transferred to a LEV RNA Cartridge. 300 μL lysis buffer were added and the rest of the extraction was performed using a Maxwell MDx AS3000 machine (Promega) following manufacturer's instructions. RNA integrity and quantity were assessed with an Experion system (Bio-Rad), and only high quality samples (RIN value ≥ 7) were selected. Random library
Experimental Factor: time
21
Experimental Factor: environmental history
sample taken during the dark
Experimental Factor: genotype
slm1 homozygous mutant