title: Wyeomyia smithii cDNA microarray (U of Oregon)
status: Public on Feb 13 2010
contact.city: Eugene
contact.first: Kevin
contact.middle: James
contact.last: Emerson
contact.state: OR
contact.address: 5289 University of Oregon
contact.department: Center for Ecology and Evolutionary Biology
contact.country: USA
contact.institute: University of Oregon
organism: Wyeomyia smithii
description: This is a spotted cDNA microarray.
  ONLY CLONES OF INTEREST (30) HAVE BEEN SEQUENCED AND IDENTIFIED.
manufacture_protocol: Construction of the cDNA Library.
  Generation of the Hybrid Line
  In order to maximize the coverage in our cDNA library of the among-population variation that exists within Wyeomyia smithii, we generated a hybrid line that includes both the nuclear and cytoplasmic genomic components of eight populations representing the latitudinal and altitudinal range of the species (Table 2).  The hybrid line was created with a four-generation crossing design (Figure 4), that, in the first three generations, created eight lines that each had a single population’s cytoplasmic component combined with all eight nuclear genomes.  These eight lines were combined in the fourth generation that then contained the cytoplasmic and nuclear genetic variation present within all eight parental populations.
  To begin each generation of crosses, larvae were synchronized into diapause by rearing individuals from the day of oviposition under short-day (Light:Dark = 8:16) conditions for at least 30 days.  At least 100 animals from each parental cross were then allowed to develop and mate under long-day (Light:Dark = 18:6), field-based conditions with a smooth, sine-wave thermoperiod that ranged from 13 to 29°C each day.
  Creation of the cDNA library
  To allow for the greatest representation of variation in the cDNA library, RNA was collected from the eight-population hybrid line.  Total RNA was extracted (TRIzol Reagent, Invitrogen) from three replicate samples of 30 III and 30 IV instar larval heads sampled at four times of day (1, 7, 13, 19 h after lights on) under both short-day (L:D = 10:14) and long-day (L:D = 18:06) conditions.  These samples (all with a 260/280 ratio of 2.0 ± 0.1) were all pooled into a total RNA pool.  One µg from this pooled RNA preparation was used to create a cDNA library using the Clontech SMART cDNA Library Construction Kit (BD Biosciences; Protocol #PT3000-1), following the manufacturer’s instructions.  Briefly, Sfi-I digested and purified cDNAs were directionally ligated and packaged into the Lamda TrplEx2 vector using Gigapack III Gold Packaging Extract (Stratagene).   The library was amplified according the Kit’s Instructions (BD Biosciences; Protocol #PT3000-1).
  The phage library was converted to a plasmid library and transformed into competent Escherichia coli (BM25.8) (Clontech protocol PT3003-1).  Individual colonies were isolated, and after overnight incubation, were used as templates for Polymerase Chain Reaction (PCR) amplification using the LD-amplimer primer pair from Clontech (forward: 5’ TCCGAGATCTGGACGAGC 3’; reverse: 5’TAATACGACTCACTATAGGG 3’) with a 5’ amine modification (IDT; /5AmMC12/).  Sixteen reactions from each 96-well PCR plate were examined using 1.0% agarose gel electrophoresis to check product sizes.  Insert sizes were between 0.25 kb and 3 kb, and a vast majority of these were single products.
  PCR reactions were precipitated (2.5 volumes 95% EtOH, 1/10 volume 3M NaOAc) and re-suspended in nanopure water.  Reactions were then dried under vacuum and each reaction was re-suspended in 10 µl 3X SSC.  The cDNA library was assessed for redundancy by sequencing 1000 random clones.  Of the 1000 sequences, 43.2% were unique suggesting that the library has a redundancy of ~60%.  As the redundancy was low for a cDNA library, no normalization was performed.
  Printing of Microarrays.
  The W. smithii microarrays were printed on silylated aldehyde (CEL-1) glass slides at the Genomics Facility of the University of Oregon.  The slides were printed, allowed to dry overnight, and then UV cross-linked at 6000J.  On the day of hybridization, slides were post-processed by immersing slides in a wash buffer (5X SSC, 0.1% SDS, 0.1 mg/mL BSA) at 42ºC for 60 minutes, and then washing the slides three times in 0.1X SSC.  The slides were then immersed in the blocking solution (2X SSC, 0.05% SDS, 2.5 mg/mL NaBH4).  After 3 washes in 0.1X SSC, slides were dried by centrifugation at 800 rpm for 5 minutes.