alias: E-MTAB-6234:15_M6_f_p
title: Illumina HiSeq 4000 paired end sequencing; RNA-seq to investigate the effect of long term exposure to fish kairomones on gene expression in Daphnia galeata
design: RNA-seq to investigate the effect of long term exposure to fish kairomones on gene expression in Daphnia galeata
library_construction_protocol: 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 strains were maintained for each of the populations, i.e. lake of origin of the sediment. After growth and treatment, the three biological replicates for two strains from one population were obtained by collecting 3 sets of 20 individuals each. The individuals collection was conducted during a short time window (between 10 AM and 1 PM) over 2 days.  All clonal lines were maintained at 18°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 at 20°C in 16:8 light:dark cycles mimicking summer conditions,  as described in the growth protocol.  For the control treatment, filtered medium  (pore size: 1.2 µm) aerated for 24 h in a fish-free tank was used.  For the fish kairomone treatment, five ide (Leuciscus idus) were placed in an aerated  tank medium for 24 hours prior to filtering. The medium was supplemented with a suspension of the algae Acutodesmus obliquus (equivalent to 2mgC/mL)  and was changed daily and the individuals fitness checked. The treatment protocol was applied during two generations (ca. 6 weeks)  before collecting individuals of the third generation born in the treatment condition. RNA was extracted by first homogenizing 20 adult individuals (whole bodies) in RNAMagic (Bio-Budget technologies). The homogenate was further mixed with chloroform and centrifuged, and the supernatant was mixed with pure ethanol and further processed with the Direct-zol RNA MiniPrep Kit (Zymo Research), following manufacturer's instructions, with an on-column DNase treatment.   Library construction for all samples was performed by Macrogen, with the TruSeq RNA Sample Prep Kit v2.
identifiers.namespace: Universitaet Hamburg
identifiers.id: E-MTAB-6234:15_M6_f_p