Mch peptide

Hippocampal neurons were cultured from embryonic day 18 C57BL/6 wild-type mice and 1 million neurons were plated on each well of a six-well plate coated with poly-d-lysine (Millipore) and laminin (Invitrogen). Neurons were maintained in Neurobasal Medium (Thermo Fisher Scientific, 21103049) supplemented with B27 (1:50 dilution; Thermo Fisher Scientific, 17504044), 12 mM glucose, glutamax (1:400 dilution; Thermo Fisher Scientific, 35050061), penicillin–streptomycin (1:500 dilution; Thermo Fisher Scientific, 15140122), 25 μM β-mercaptoethanol and 20 μg ml⁻¹ insulin (Sigma-Aldrich, I9278). To prevent overgrowth of glia, neuron cultures were treated with 10 μM 5-fluoro-2′-deoxyuridine (Sigma-Aldrich, F0503) after 3 d. Neurons were treated with control vehicle (H₂O) or 1 µM MCH peptide (H-070-47, Phoenix) for 4 h, and collected for immunoblotting or RNA extraction on day in vitro (DIV) 12–14.

For bulk RNA-seq, two independent cultures of hippocampal neurons were used. In each culture, we treated two wells with control vehicle (H₂O) and two wells with 1 µM MCH peptide (H-070-47, Phoenix) for 4 h. RNA was extracted using the RNeasy Mini Kit (Qiagen 74104). RNA purity (260/280 and 260/230 ratios) and integrity were assessed using Nanodrop ND-1000 (Nanodrop Technologies) and an Agilent 2100 Bioanalyzer with High Sensitivity chips (Agilent Technologies) and a Qubit 3.0 Fluorometer (Life Technologies), respectively. RNA integrity values of the samples ranged from 7.9 to 9.3 (median, 8.6). Library preparation from total RNA extraction and sequencing were performed at the VIB Nucleomics Core. Briefly, 1 µg of total RNA extract per sample was enriched for mRNA molecules using poly-T oligonucleotide-attached magnetic beads. The enriched poly-A mRNA species was subjected to fragmentation and reverse transcription using random primers, and the subsequent library processing was done using the standard Illumina TruSeq Stranded mRNA Sample Prep Kit (protocol version 15031047 rev. E). Libraries were sequenced using an Illumina NovaSeq 6000 instrument at an average depth of approximately 31 million reads. Raw reads were pre-processed, mapped and quantified against the GRCm38 Mus musculus genome using the nf-core/rnaseq pipeline (v3.0; 10.5281/zenodo.1400710).