RNA-sequencing of preoptic area leptin receptor-expressing neurons from heat acclimated LepRCreHTB mice
RNA-sequencing of preoptic area leptin receptor-expressing neurons from heat acclimated LepRCreHTB mice
Individual adult mice LeprCreHTB, acclimated and non-acclimated, (P77 ± 5) were anesthetized with isoflurane and decapitated, the brain was immediately removed and submerged in ice-cold artificial of POALepR neurons involved in heat acclimation cerebrospinal fluid (ACSF). 3 brains were sectioned at the same time on a vibratome (Leica VT1200S) in a slicing chamber containing ice-cold ACSF (in Mm; 1.2 NaH2PO4xH2O, 2.5 KCl, 20HEPES, 25 glucose; 30 NaHCO3, 93 NMDG (n-methyl-glucamine), 5 Na-Ascorbate, 3 Na-pyruvate, 12 N-acetylcysteine, 10 MgSO4x7H2O, 0.5 CaCl2), constantly bubbled with carbogen. Brain slices of 250 μm thickness, containing the OVLT and MnPOA, were transferred to a Petri dish containing ACSF. We implemented the neuron isolation protocol described in Moffit et al. 2018. The regions of interest were micro dissected under a dissecting microscope and transferred to a small Petri dish containing 3ml of Papain mix consisting of Hibernate mix (Hibernate A medium (Invitrogen A1247501), 1xGlutamax (Gibco 35050-038), 0.8mM Kynurenic acid (Sigma K3375-5G), 0.05 mM AP-V (HelloBio HB0225), 0.01 mM Rock inhibitor Y-27632 (HelloBio HB2297), 1 mM B27 (Invitrogen 17504001), 5% Trehalose (Sigma T9531-10G)) and 8U/ml papain (Sigma P4762), 100U/ml DNAseI (Worthington über Cell-Systems LK003172), 0.005U/ml Chondroitinase ABC (Sigma C3667-5UN), 0.07% Hyaluronidase (Sigma, H2126), 0.001 mM NaOH where the tissue was cut in smaller pieces. The dissected tissue pieces were transferred together with the Papain mix into a 2ml tube at 37˚C to incubate while gently shaking (700 rpm) for 2h. After incubation, with the tissue pieces sitting at the bottom of the tube, the papain solution was pipetted out of the tube and exchanged with the hibernate mix containing 0.1mg/ml ovalbumin and centrifuged for 1 min at 300 g. Supernatant was removed, and hibernate mix was added to the tissue pieces which were further dissociated into single cells by gentle trituration through Pasteur pipettes with fire-polished tip openings of 600-μm, 300-μm, and 150- μm diameter. The cell suspension was centrifuged at RT at 300 g, for 10 minutes, and the supernatant was removed and exchanged with 500mul of Hibernate A medium.  FACS sorting Resuspended cell material was passed through a 20um filter. The cell suspension was stained with propidium iodide (PI BD Pharmingen, Cat 51 66211E, 2ml - 50ug/ml) to exclude the dead cells before the FACS analysis. FACS sorting was performed on BD FACS Aria II Flow Cytometry Cell Sorter using the “purity” sorting mode. FACS populations were arbitrarily chosen to select cells with low PI and high GFP fluorescence but the criteria were kept the same for each sorting round. Cells were FACS sorted into bulks of GFP+ and GFP directly into the RLT buffer (QIAGEN RNeasy Micro Kit 74004), immediately frozen on the dry ice, and stored at -80˚C.  Samples were further processed in a maximum of 1 month from the isolation day, by using the column purification method with QIAGEN RNeasy Micro Kit according to the manufacturer's instructions. Samples were stored at -80 ˚C until further processing.   Samples were further processed in a maximum of 1 month from the isolation day, by using the column purification method with QIAGEN RNeasy Micro Kit according to the manufacturer's instructions. Samples were stored at -80 ˚C until further processing.  RNA integrity and concentration of each sample were assessed by Agilent Bioanalyzer Nano 6000 chip (Agilent technologies) and QUBIT2 fluorometer measurement (Thermofisher Scientific) by manufacturer's instruction. I used the Smart-Seq2 protocol for the cDNA library preparation (all processing performed at Gene Core EMBL, Heidelberg). 1ul of each bulk sample RNA was processed for the reverse transcription (Superscript IV, ThermoFisher Scientific, cat. no. 18090010) followed by 18 cycles of PCR amplification, library tagmentation via Tn5 transposase (Tn5 was produced in-house by Protein Expression and Purification Core Facility, EMBL Heidelberg), sample barcoding (Illumina primers) and final enrichment by 12 cycles of PCR.