Name: SAN_LA_L_2_s
Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: PolyA
Layout: SINGLE
Construction protocol: All adult samples were collected from flies aged between 2-10 days. Antenna were collected by flash freezing flies in liquid nitrogen and agitating them over a sieve connected to a collection dish. Antenna were selected from the collection dish using a pipette under a dissecting scope. Forelegs and proboscis with maxillary palps were collected from individual files using forceps and microscalpels under a dissecting scope. Third instar larva were collected from vials by floating them in 75% sucrose water and washed. Larva heads were removed under a dissecting scope using a microscalpel. For each replicate, ~10 third instar larval heads, ~25 proboscis, ~50 legs, ~5 ovipositors and ~100 antennae were collected. After sorting sexes on C02, all flies were left in food vials for at least 24h before the dissections. All adult samples were collected from flies aged between 2-10 days. Antenna were collected by flash freezing flies in liquid nitrogen and agitating them over a sieve connected to a collection dish. Antenna were selected from the collection dish using a pipette under a dissecting scope. Forelegs and proboscis with maxillary palps were collected from individual files using forceps and microscalpels under a dissecting scope. Third instar larva were collected from vials by floating them in 75% sucrose water and washed. Larva heads were removed under a dissecting scope using a microscalpel. For each replicate, ~10 third instar larval heads, ~25 proboscis, ~50 legs, ~5 ovipositors and ~100 antennae were collected. After sorting sexes on C02, all flies were left in food vials for at least 24h before the dissections. Fly strains were reared on a standard yeast/cornmeal/agar medium supplemented with Carolina 4-24 Formula and maintained in a 12:12 hr light:dark cycle at 25 degrees. Adults between 2 to 10 days old were sex-sorted on CO2 at least 24h before the dissections. Dissected tissues were homogenized in 200ul of Trizol (Invitrogen) using a Precellys24 (6800rpm, 2x30s with 10s breaks ; Bertin Technology) followed by a standard Trizol RNA extraction. The final mRNA concentration was measured using a DeNovix Ds-11 FX spectrophotometer. mRNA libraries were prepared using KAPA Stranded mRNA-seq Kit (Roche) following manufacturer’s instructions (Version 5.17). Briefly, 500ng of total RNA diluted in 50ul of RNAse-free water was first placed on supplied mRNA capture magnetic beads to allow the isolation of mature, polyadenylated, mRNA which was subsequently fragmented to a size of 100-200bp. Double-strand cDNA was then synthesized, marked by A-tailing and barcoded with 2.5ul of TruSeq RNA UD Indexes (Illumina). SPRI select beads (Beckman Coulter) were used for cleanup.