title: GSM9117443: PVH nuclei, male, fed, sample 8, drop 2, RT-rxn A, lib. 1; Mus musculus; RNA-Seq
library_construction_protocol: C57BL/6J or Sim1-Cre::R26-LSL-EGFP-L10a mice aged 6-12 weeks were sacrificed between 9 am – 12 pm by rapid decapitation immediately after removal from the home cage. Brains were rapidly extracted and chilled in DMEM/F12 media slush. Next, brains were placed ventral side up in a chilled stainless steel brain matrix (Roboz Surgical Instrument Co.: SA-2165) and 1 mm coronal sections of the hypothalamus were collected. The PVH was then microdissected under a fluorescent stereoscope and frozen. For each sample preparation, 4-6 male or female mice were pooled. 4-6 tissue dissections were placed in a dounce homogenizer with 1 mL cold (4C) Lysis Buffer containing 10 mM trisHCl pH 8 (Sigma-Aldrich), 250 mM Sucrose (Sigma-Aldrich), 25 mM KCl, 5mM MgCl2 (Sigma-Aldrich), 0.1% Triton x100 (Sigma-Aldrich), 0.5% RNasin Plus RNase Inhibitor (Promega), 0.1 mM Dithiothreitol (DTT) (Sigma-Aldrich) in UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific). After douncing for 20 times, the solution was filtered through a sterile 20 µm Cell Strainer (pluriSelect), collected in 1.5 mL DNA LoBind® Tubes (Eppendorf), and centrifuged for 10 min at 900 g (rcf) at 4 C. The "slow sedimenting" component (debris and membranes) was aspirated and discarded while the "fast sedimenting" component (nuclear fraction) was gently resuspended in a 1 mL of Working Solution containing 1X pH 7.4 RNase free PBS (Thermo Fisher Scientific), 0.01% Albumin Bovine Serum (BSA) (Sigma-Aldrich), 0.5% RNasin Plus RNase inhibitor (Promega) in UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific). Nuclei were kept on ice while transferred to the BNORC Functional Genomics and Bioinformatics (FGB) Core for DroNc-seq assay. DroNc-seq was performed as per Habib et al. 2017, with minor modifications. Briefly, nuclei stained with Hoechst 33342 (ThermoFisher, cat. R37605) were counted on a hemocytometer and diluted in NSB to ~250,000 nuclei/ml. Barcoded beads (Chemgenes, Cat # Macosko-2011-10) were size-selected using a 40 μm strainer, diluted to 350,000 per ml, and loaded onto 70 μm wide and 75 μm deep microfluidic device (Nanoshift). The nuclei and barcoded bead suspensions were loaded and run at 35 ml/hr each, along with carrier oil (BioRad Sciences, Cat # 186-4006) at 200 ul/min, to co-encapsulate single nuclei and beads in ~75 μm drops (vol. ~ 200 pl) at 4,500 drops/sec and double Poisson loading concentrations. The microfluidic emulsion was collected into 50 ml Falcon tubes for 10-25 min each and placed on ice 2h before droplet disruption. Individual 200µl reverse transcription (RT) reactions were performed on up to 90K beads. After further exonuclease digestion, aliquots of 800-5K beads were PCR amplified for 10 cycles, and PCR products were pooled in batches of 4 wells or 16 wells for library construction. Purified cDNA was quantified, and 550 pg of each sample was fragmented, tagged, and amplified in each Nextera reaction. Libraries were sequenced on the Illumina NextSeq 500 using between 1.6–1.7 pM and 0.3 μM Read1CustSeqB (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC).