Magrack 6

This protocol was adapted from³¹ (Fig. 4). Third-instar larvae of the following genotype: w; Orco-Gal4; UAS-GFP-Msp300^KASH were used for OSN nuclei isolation. Larvae were placed in PBS during sample collection. A pair of surgical scissors was used to dissect out the dorsal organ of the larvae. Larval mouth hooks provided a visual landmark during the dissection. Incisions were made so as to exclude larval brains from the final sample collection. Dissected samples were stored in cold PBT (PBS plus 0.1% Tween-20 (VWR #0777)). Samples were homogenized using a mortar and pestle. Pre-isolation samples were collected to determine nuclear yield, nuclei integrity, and to determine transcript levels of target genes prior to nuclei isolation. Affinity based isolation of nuclei was performed as described in³¹ . Briefly, GFP-Msp300^KASH tagged OSN nuclei were pulled down using a Chicken anti-GFP antibody (Invitrogen #PA1–9533) bound to magnetic Dynabeads Protein G (Invitrogen #10003D). Antibody-bound beads and homogenate were placed in a magnetic rack, MagRack6 (GE #28948964) for 2 minutes to allow the magnetic beads to bind to the magnet. Homogenate containing the unbound nuclei fraction was removed (Fig. 4c). Following 3x washes with a Wash buffer, post-isolation samples were suspended in 350 µl RLT buffer and stored at −20C until they were subjected to the RNA extraction protocol.

Magnetized Nanotrap® CN3080 particles from Ceres Nanoscience (www.ceresnano.com) were used. These particles contain Reactive Red 120 Dye as the chemical bait. Nanotraps were provided from Ceres either as a sterile solution of particles at 5 mg/mL in buffer, or as a lyophilized pellet pre-aliquoted in a tube (0.25 mg Nanotraps). The estimated size of these particles is 400–900 nm decorated with 80–120 nm magnetic spheres. In order to separate magnetic Nanotraps from a solution, the GE MagRack 6 was used. The magnetic bar was placed next to the microcentrifuge tube for approximately 5 min in order to “pull-down” the particles.