Illumina HiSeq 3000 sequencing; RNA-seq of resting plasmatocytes from Drosophila melanogaster third instar larvae
RNA-seq of resting plasmatocytes from Drosophila melanogaster third instar larvae
FLY RAISING Flies were raised and maintained on standard cornmeal food at 25°C or room temperature. For experiments on wild type OrR, adult fly collections were left to lay eggs on apple juice agar plates with yeast paste overnight at 25°C. After 24h hatched larvae were washed of the plate and further larvae were allowed to hatch for 2h. Then, larvae were picked of the plate and transferred into 36mm food tubes with yeast paste at a density of 120 larvae per tube and raised at 25°C. The resulting larval collection tubes would reach 3rd instar wandering stage around 4 days after hatching, at which stage plasmatocytes were extracted.  PLASMATOCYTE ISOLATION FOR DIRECT RNA EXTRACTION For plasmatocyte extraction and RNA isolation, treated or untreated 3rd instar wandering larvae were collected from food tubes and washed in PBS. At the same time Schneider's medium with FCS with 10 mM N-Acetyl-L-Cysteine was prepared in a well of a 24-well tissue culture plate (Sarstedt). Larvae were dried on paper towels and transferred to the tissue culture well with medium. Then they were ripped by holding the head with forceps (Dumont) and with another forceps ripping open the cuticula along the body of the larvae without disrupting the gut. In this ways 80 larvae were bled for each sample. The larval carcass was then removed and the plasmatocytes were allowed to attach for 10-15 minutes. Afterwards, the plasmatocytes were washed 4 times with PBS, then all supernatant was aspirated, they were lysed in 94.5 µl RNAdvance lysis buffer and processed immediately.  RNA ISOLATION BY RNADVANCE RNA was isolated from freshly lysed plasmatocytes samples using the RNAdvance cell v2 kit following manufacturer's instructions using a DynaMag-2 magnetic rack. DNase digestion was performed as suggested in the manual using Ambion DNase. Resulting RNA samples were eluted in 20 µl ultra-pure water and stored at -80°C. RNA was isolated from freshly lysed plasmatocytes samples using the RNAdvance cell v2 kit following manufacturer's instructions using a DynaMag-2 magnetic rack. DNase digestion was performed as suggested in the manual using Ambion DNase. Resulting RNA samples were eluted in 20 µl ultra-pure water and stored at -80°C. Libraries were generated by the Max Planck Genome Centre (Cologne). Libraries were stranded, from polyA enriched RNA.