alias: GSM4416969
title: GSM4416969: OT01_P3_G07; Mus musculus; RNA-Seq
library_construction_protocol: OT-2A-Flp::fdGFP mice were weaned at P21 and given i.v. injections of FluoroGold on that and the following day. At P23 mice were sacrificed. Brains were rapidly removed and 250 µm thick coronal slices (n = 7 mice) containing the paraventricular nucleus of the hypothalamus were sectioned using a Leica VT-1200s vibrating microtome in ice-cold ACSF solution containing the following (in mM): 124 NaCl; 2.5 KCl; 1.2 NaH2PO4; 24 NaHCO3; 5 HEPES; 13 glucose; 2 MgSO4'; 2 CaCl2, oxygenated with carbogen gas (95% O2 and 5% CO2) to pH 7.3 - 7.4. To microdissect the PVN, slices were placed in a petri-dish containing ice-cold, oxygenated ACSF solution and the PVN was identified using the 3rd ventricle and other structural markers. Following dissection, tissue was placed into 2.6 mL equilibrated Papain DNase-I solution. The dissociation protocol was similar to Chevée et al., 2018, itself an adaptation from the trehalose-enhanced neuronal isolation protocol using the Worthington Papain Dissociation System (Worthington Biochemical Corporation). The following modifications from Chevée et al., 2018 were made: a single low speed centrifugation (300xg for 5 min) was performed after dissociation, and the pellet was resuspended in 250 µL media (DMEM, 5% trehalose (w/v), 25 µM AP-V, 0.4 mM kynurenic acid, 6 µL of 40 U/µl RNase inhibitor) and 250 uL of EBSS#2 (EBSS, 25 mM AP-V, 100 mM Kynurenic acid, ovomucoid protease inhibitor with BSA, DNase-I, 5% w/v Trehalose, 40 U/µl RNase inhibitor) at room temperature. Following resuspension, single-cell suspensions were introduced into a FACS machine (Beckman Coulter MoFlo Cell Sorter).     Neurons were sorted based on fluorescence (GFP+/FG- or GFP+/FG+) directly into individual wells of a 96-well plate containing 2 µL Smart-Seq2 lysis buffer + RNAase inhibitor, 1µL oligo-dT primer, and 1 µL dNTPs(48). Three plates were collected across 2 separate dates (Plate 1: n = 1 mouse, 32 GFP+/FG- neurons and 47 GFP+/FG+ neurons; Plate 2: n = 1 mouse, 32 GFP+/FG- neurons and 33 GFP+/FG+ neurons; Plate 3: n = 5 mice, 25 GFP+/FG- neurons and 48 GFP+/FG+ neurons). Following the sort, plates were briefly spun-down in a table-top microcentrifuge and immediately placed on dry ice. Single-cell lysates were kept at -80°C until cDNA library preparation. Library preparation and amplification of single-cell samples was performed using a modified version of the Smart-Seq2 protocol as described in Cheveé et. al 2018. Individual libraries were quality controlled, pooled, and sequenced on two lanes of an Illumina HiSeq 2500 sequencer to an average depth of 1,424,499 ±77,731 paired-end 50bp reads per neuron.
identifiers.namespace: GEO
identifiers.id: GSM4416969