title: Illumina HiSeq 2000 sequencing; Early transcriptional events linked to induction of diapause revealed by RNAseq in larvae of drosophilid fly, Chymomyza costata.
design: Early transcriptional events linked to induction of diapause revealed by RNAseq in larvae of drosophilid fly, Chymomyza costata.
library_construction_protocol: A wild-type (Sapporo) strain of Chymomyza costata (Zetterstedt, 1838) (Diptera: Drosophilidae) originally collected in Sapporo, Hokkaido, Japan, in 1983 was used. Insects were cultured at constant temperature of 18 degree C in incubators MIR154 (Sanyo Electric, Osaka, Japan) on an artificial diet as described by (Kostal et al., 1998). Developmental destiny of larvae was programmed using two different photoperiodic regimes: a long day regime (LD, 16 hour  light : 8 h dark ) at which all larvae of both strains continue direct development (i.e. pupariate, pupate and metamorphose to adults), and a short day regime (SD, 12 hour  light : 12 hour  dark) that induces larval diapause in all individuals of Sapporo strain. In order to switch the developmental destiny from direct development to diapause, the LD-reared wild-type larvae were transferred to SD conditions (T, transfer) on day 3 of their 3rd larval instar (i.e. approximately when 15-day-old since the egg deposition). The transferred larvae experienced 4h advanced light OFF time (dusk) on day 15. All wild-type strain larvae respond to such transfer by switching the developmental destiny from direct development to diapause. All experimental insects came from synchronized eggs (laid within one day) and a second developmental synchronization took place during the 2nd to 3rd instar transition that means three days prior to sampling. All samples were taken on day three of 3rd instar when the larvae reach their maximum sensitivity to photoperiodic signal. The samples were taken at two Zeitgeber times (Zt) in each photoperiodic regime (LD, SD, T). Three biological replicates were taken at each Zt, and each replicate consisted of a pool of 10 larvae. Night samples were taken under dim red light. Larvae were collected to 400ul of ice cold RiboZol RNA Extraction Reagent (Amresco, Solon, OH, USA) in 1.5 ml microvial, immediately cut to small pieces by scissors and stored at -80 degree C until next processing.  The total RNA was extracted from whole larvae using the RiboZol RNA Extraction Reagent. Pellet of total RNA was dissolved in 20 ul of DEPC-treated water and an aliquot of 5 ul was taken for total RNA quality assessment on denaturing agarose gel and concentration measurement using Cary50 UV-VIS spectrophotometer (Varian, Palo Alto, CA, USA). The total RNA concentrations were levelled exactly to 0.5 ug/ul and the samples were either sent to the EMBLGenomics Core Facilities (GeneCore, Heidelberg, Germany) for cDNA library production and Illumina RNAseq or used for qRT-PCR validation of RNAseq results The cDNA libraries were prepared from the samples of Sapporo strain larvae using Covaris S2 (Covaris, Woburn,Massachusetts, USA) for fragmentation aiming for an insert size of about 150 nt and TruSeq RNA sample prep kit (Illumina, San Diego, California, USA)
attributes.tag: Experimental Factor: stimulus
attributes.value: Photoperiod light 16/ dark 8 then light 12/ dark 12
attributes.tag: Experimental Factor: growth condition
attributes.value: Larvae transferred from long day to short day