title: Cell-type–resolved RNA-seq reveals molecular engrams of sexual experience in Drosophila neuromodulatory neurons
design: Cell-type–resolved RNA-seq reveals molecular engrams of sexual experience in Drosophila neuromodulatory neurons
library_construction_protocol: About 100 adult male flies collected from 3-4 days F1 generation of NPFR GAL4_driver X UAS_unc84_2XGFP_ reporter were subjected to sexual experience for 2 days and flash frozen. Fly heads were separated from bodies on dry ice and stored at –80 °C until RNA extraction. Flies were kept in the incubator at 25°C, ~50% humidity, and light/dark of 12:12 hours Males and females were collected within 2 h of eclosion on CO2, 3-4 days before courtship conditioning. Males were collected into narrow glass vials (VWR culture glass tubes 10X75mm) containing food and kept single housed until the conditioning. To generate mated females for the experiment, mature males were added to the females ~16 h before the experiment. The mated females were separated from the males on the morning of the conditioning. During the conditioning, the temperature was kept at about 25°C, and humidity was ~55%. Generation of rejected males: Individual males were placed with mated females for 3 one-h conditioning trials (separated by 1-h rests) a day for two or four consecutive days. Females were removed after each trial. Males from the rejected cohort that managed to mate and males from the mated cohort that did not end up mating during all sessions were discarded. At the end of each session, the female fly was removed, and the males that experienced rejection were kept in the original vial for one hour of rest. Males were monitored every 10 minutes to ascertain lack of mating, and when mentioned the number of males exhibiting courtship action during training sessions was documented.  The NuGEN RNAseq v2 (7102-32) kit was used to prepare cDNA from the INTACT purified RNA Cell type specific labeled nuclei were isolated using the INTACT method (Isolation of Nuclei Tagged in A specific Cell Type technique). Frozen heads were homogenized using 9ml of homogenization buffer (20mM β-Glycerophosphate pH7, 200mM NaCl, 2mM EDTA, 0.5% NP40 supplemented with RNAase inhibitor,10mg/ml t-RNA, 50mg/ml ultrapure BSA, 0.5mM Spermidine, 0.15mM Spermine and 140ul of carboxyl Dynabeads -270 (Invitrogen: 14305D) was added to each sample. The heads were filtered on ice by a series of mechanical grinding steps followed by filtering the homogenate using a 10um Partek filter assembly (Partek: 0400422314). After removing the carboxyl-coated Dynabeads using a magnet, the homogenate was filtered using a 1um pluriSelect filter (pluriSelect: 435000103). The liquid phase was carefully placed on a 40% optiprep cushion layer and centrifuged in a 4oC centrifuge for 30min at ~2300Xg. The homogenate/Optiprep interface was incubated with an anti-GFP antibody (Invitrogen: G10362) and protein G Dynabeads (Invitrogen: 100-03D) for 40 minutes at 4oC. Beads were then washed once in NUN buffer (20mM β-Glycerophosphate pH7, 300mM NaCl, 1M Urea, 0.5% NP40, 2mM EDTA, 0.5mM Spermidine, 0.15mM Spermine, 1mM DTT, 1X Complete protease inhibitor, 0.075mg/ml Yeast torula RNA, 0.05Units/ml Superasin). Bead-bound nuclei were separated using a magnet stand and resuspended in 100ml of RNA extraction buffer (Picopure kit, Invitrogen # KIT0204), and RNA was extracted using the standard protocol. Library preparation was performed using the SPIA - NuGEN Encore Rapid DR prep kit.