Strand-specific RNAseq - Chlorella vulgaris - Autotrophy - 72h after inoculum - Replicate 2
Chlorella vulgaris strain CPCC 90 was grownĘon synthetic modified BBM (Bold's Basal Medium) medium (Stein-Taylor, 1973 - ISBN 0-521-29747-8). Cells were cultivated in batch mode in a 7-L bioreactor (BioFlo 115 - New Brunswick Scientific, USA) with 5 L of culture medium. The culture was initiated Ęwith a biomass concentration of 0.2 gáL-1 (exponential growth phase) at 26ź C, aeration rate of 1 vvm and continuous agitation at 150 rpm. Three different cultivation conditions were carried out in sequence in the same batch culture starting with autotrophy (first 96 hours), followed by mixotrophy (96.05 hours up to 128 hours) and heterotrophy (from 128.05 hours to 160 hours). For autotrophic and mixotrophic growth, filter-sterilized air was enriched with CO2 to 1% (v/v) and illumination was provided by sixteen 40 W fluorescent lamps, with a constant light intensity of 209 ľmol.m-2.s-1. BBM was supplemented with 10 gáL-1 glucose for heterotrophic and mixotrophic conditions. For heterotrophic conditions, the light and CO2 supply was interrupted. A volume of 2 mL of cell culture were harvested by centrifugation and cells in the pellet were disrupted by 1 minute maceration in a Mini Beadbeater (Biospec Product) using 1.0 mm glass beads. Total RNA of three independent replicates for each sample was extracted using Trizol (Invitrogen, USA) according to manufacturerŐs instructions. RNA quality and concentration was assessed by gel electrophoresis, NanoDrop (Thermo Fisher Scientific) and Bioanalyzer (Agilent Technologies). Only RNA samples with a minimum RNA Integrity Number (RIN) of 8 were further processed. Libraries were produced using TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturerŐs instructions. Clusters were made on c-Bot (Illumina) and paired-end sequencing was carried out on a Hi-Seq 2500 (Illumina) using TruSeq SBS Kit v3 Đ HS (Illumina). Samples were sequenced at the LACTAD Facility (University of Campinas, Campinas, Brazil).
P4E3