Agilent bioanalyzer pico chips

We carefully staged control (phm>w¹¹¹⁸) and phm>séan-RNAi (VDRC #100854) larvae at the L2/L3 molt, and we dissected ring glands at 44 ± 0.5 hr after the molt. These samples were then transferred to ice-cold TRIzol reagent (Thermo Fisher Scientific). For each sample, the lysates of 20 ring glands were vortexed at room temperature for 5 sec, briefly spun down, and stored at −80° until use. Total RNA was isolated by NucleoSpin RNA (Macherey-Nagel, Düren Germany), quantified by RiboGreen Quanti Kit (Thermo Fisher Scientific), and RNA integrity was analyzed by Agilent Bioanalyzer Pico chips. We used 100 ng of total RNA per sample as input for cDNA library synthesis. Each genotype was analyzed by two independent biological replicates for analysis. The Encore Complete RNA-Seq library systems (NuGEN Technologies, San Carlos, CA) were used to produce the cDNA libraries for next-generation sequencing, following the manufacturer’s instructions. The cDNA libraries resulting from the Encore RNA-Seq systems were pooled together in equal concentrations for sequencing at McGill University and the Génome Québec Innovation Centre (Montréal, Canada). We normalized raw data with ArrayStar 4.0 (DNASTAR), and data were analyzed by ArrayStar 4.0, Access (Microsoft, Redmond, WA), and the Database for Annotation, Visualization and Integrated Discovery (DAVID) (Huang et al. 2009 (link)).

Hypothalamic sections of the ARC were prepared on a cryostat at 14 µm thickness from approximately −4.52 to −2.30 mm relative to Bregma (Paxinos and Watson, 1998 ). Sections were collected onto RNase-free membrane-coated slides (PALM Microlaser Technologies) that had been baked at 200°C for 4 h and UV cross-linked for 30 min. Within 24 h of sectioning, sections were placed for 30 s each time in 95%, 75% and 50% ethanol for rehydration. Sections were stained with 1% Cresyl Violet stain (Ambion) for 1 min, dehydrated in graded ethanol concentrations (50%, 75% and twice in 100% for 30 s each time), placed in HistoClear (National Diagnostics) for 5 min and air dried. LCM was performed using a PALM MicrolaserSystem (Fig. 4A). Following microdissection, the captured cells were kept in RNAlater (Ambion). Total RNA was isolated from LCM samples using the RNAqueous Micro RNA extraction kit (Ambion) in accordance with the manufacturer's protocol. The quality and quantity of the RNA samples was determined using Agilent BioAnalyzer PicoChips (Agilent Technologies). Total RNA was isolated from E16.5 fetal heads and neonatal and adult (3 months of age) hypothalami as previously described (Zaibi et al., 2010 (link)) and analysed using a NanoDrop ND1000 (Thermo Fisher Scientific).