Illumina HiSeq 2000 sequencing; Population transcriptomics in Daphnia galeata
Population transcriptomics in Daphnia galeata
Sample 64_s
Lake sediment was sampled and sorted manually in order to obtain resting stages of Daphnia, so called ephippia.  These ephippia were incubated in artificial medium (ADaM) at 20°C in 16:8 light:dark cycles mimicking summer conditions, and the individuals hatching from these ephippia were used to establish clonal lines, all individuals in these lines having the same genotype.  Six different genotypes were maintained for each of the populations, i.e. lake of origin of the sediment. After growth and treatment, the three biological replicates for each genotype were obtained by collecting 3 sets of 30 individuals each. The individuals collection was conducted during a short time window (between 10 AM and 1 PM) over 4 days. All clonal lines were maintained at 20°C in 16:8 light:dark cycles mimicking summer conditions. They were fed ad libitum every other day with a suspension of the algae Acutodesmus obliquus. All clonal lines were maintained for one week prior to collection and RNA extraction at 20°C in 16:8 light:dark cycles mimicking summer conditions, as described in the growth protocol. RNA was extracted by first homogenizing 30 adult individuals (whole bodies) in Trizol (Invitrogen). The homogenate was further mixed with chloroform and centrifuged, and the supernatant was mixed with pure ethanol and further processed with the Qiagen RNeasy kit, following manufacturers instructions, with an on-column DNase treatment. Library construction for all samples was performed by GATC Biotech AG, Konstanz, Germany, following proprietary protocols. Total RNA was treated as follows: poly(A)+ RNA isolation, mRNA fragmentation, first strand cDNA synthesis with random primers, 2nd strand synthesis, Adapter ligation and pooling of tagged libraries. The library prep was based on protocols for New English Biolab products.