title: Illumina HiSeq 2000 sequencing; RNA-Seq analysis of a Reciprocal Transplant experiment for two strains of Spodoptera frugiperda fed on rice or corn, in laboratory and natural populations
design: RNA-Seq analysis of a Reciprocal Transplant experiment for two strains of Spodoptera frugiperda fed on rice or corn, in laboratory and natural populations
library_construction_protocol: Spodoptera frugiperda wild larvae were collected in Florida and Georgia between September, 18th and September, 25th 2015 in three different field locations. One sweet corn field in Citra (Marion County, Florida), one volunteer corn in Tifton, (Tift County, Georgia) and one pasture grass field in Jacksonville (Duval County, Florida). In corn fields, plants were cut and larvae collected in situ. In the pasture grass field, collections were made using a sweeping net. After confirming their identification as Spodoptera frugiperda according to LepIntercept (http://idtools.org/id/leps/lepintercept/frugiperda.html), larvae were placed in individual plastic cups with cut leaves (either corn or grass) as a food source and brought back in a cooler to the laboratory after a few hours of collection. Once in the laboratory, larvae were sorted according to stage. Stages were measured according to the chart in Supplementary Figure 18, where the width of the cephalic capsule should match the width of the line for each stage. This chart has been determined based on rearing conditions of lab strains in Montpellier and confirmed with a similar chart based on the rearing of lab strains in Gainesville, Florida. L4 larvae were sacrificed with a razor blade and immediately placed in a screw-cap 2ml tube containing 1ml of RNAlater (Sigma; R0901). Younger larvae were placed again in individual cups with fresh cut leaves and were grown in a 25°C incubator until they reached the L4 stage. The reciprocal transplant (RT) experiment consisted in controlled infestations of corn and rice plants with first instar larvae in 8 insect rearing cages (32.5 x 32.5 cm) covered by an insect-proof veil to prevent contaminations and escapes in the incubator (24°C, 16h:8h L:D cycle and 70% R. H.). The RT experiment was conducted in the same incubator for four modalities: 1) corn plants infested by sf-C (native condition); 2) rice plants infested by sf-C (alternative condition); 3) corn plants infested by sf-R (alternative condition); 4) rice plants infested by sf-R (native condition). We realized two replicates by modality. Each cage contained four corn or rice pots, which were changed before the 4th larval instar and each day after this instar until the pupation. We deposited in each cage 80 larvae hatched the morning of the experiment initiation (larvae obtained from several egg-masses provided from breeding). Two generations have been conducted on plants; during the first generation we measured life history traits (LHT) for each strain in native and alternative conditions and during the second generation, the larvae had been sampled at 4th larval instar for a RNA-Seq analyzes. The reciprocal transplant (RT) experiment consisted in controlled infestations of corn and rice plants with first instar larvae in 8 insect rearing cages (32.5 x 32.5 cm) covered by an insect-proof veil to prevent contaminations and escapes in the incubator (24°C, 16h:8h L:D cycle and 70% R. H.). The RT experiment was conducted in the same incubator for four modalities: 1) corn plants infested by sf-C (native condition); 2) rice plants infested by sf-C (alternative condition); 3) corn plants infested by sf-R (alternative condition); 4) rice plants infested by sf-R (native condition). We realized two replicates by modality. Each cage contained four corn or rice pots, which were changed before the 4th larval instar and each day after this instar until the pupation. We deposited in each cage 80 larvae hatched the morning of the experiment initiation (larvae obtained from several egg-masses provided from breeding). Two generations have been conducted on plants; during the first generation we measured life history traits (LHT) for each strain in native and alternative conditions and during the second generation, the larvae had been sampled at 4th larval instar for a RNA-Seq analyzes. We weighed the pools and crushed them in liquid nitrogen to obtain a fine powder, which was placed in TRIzol® Reagent (Invitrogen) and stored at -80°C. After collection of samples in all experimental set-ups, total RNA was extracted using a TRIzol® Reagent, according to the manufacturer's RNA protocol. To remove contaminating DNA from RNA preparations, we used DNase from TURBO DNA-free™ Kit (Ambion). qRNA quantity and quality were assessed using a Bioanalyzer and 1 µl of total RNA from each sample of three larvae. The pool of three larvae, having a good quality and quantity, were pooled to obtain 6 samples corresponding to the 2 biologic replicates of the 4 experimental set-ups. Library preparation is done using Illumina's TruSeq Stranded mRNA Sample Preparation kit. It consists of 7 steps: 1. The selection of polyadenylated RNAs on magnetic magnesium oligo (dT); 2. Chemical fragmentation of the selected RNAs 3. The synthesis of the first strand of cDNA (random primers + SuperScript II) in the presence of acetoincin D in order to block the synthesis of the second strand; The synthesis of the second strand of cDNA by replacing the dTTP with dUTP in order to block the synthesis of the second strand during the PCR amplification step; Adding a base To these extremes 6. The ligation of adpatators to these ends.7. A PCR amplification step. This step also allows for an election since only the fragments bearing the two types of adapters (cf step 6) will be amplified. The validation of the libraries is performed qualitatively and quantitatively on an Agilent DNA 1000 chip as well as by qPCR (Applied Biosystems). 7500 with SYBR Green chemistry).
attributes.tag: Experimental Factor: strain
attributes.value: sf-C
attributes.tag: Experimental Factor: environmental history
attributes.value: laboratory strain
attributes.tag: Experimental Factor: diet
attributes.value: rice