title: RNA-Seq of mus musculus: cell cultured cortical neurons
design: Neurons were obtained from the embryonic brain at 17-19 days gestation from C57BL/6 mice. Briefly, pregnant female mice were deeply anesthetized with isoflurane until unresponsive, then killed by cervical dislocation. Embryos were removed from the uterus under sterile conditions. The cortex and hippocampi were isolated from embryos, cut into fine pieces, and triturated with a plastic pipette to dissociate. Dispersed cells were seeded onto high molecular weight PDL (70-150 kDa) coated cell culture plates at 80,000 cells/cm2 for 96 well plates or 120,000 cells/cm2 for coverslip-containing 24 well plates and maintained in a sterile incubator at 37C, 5% CO2. Standard media (high glucose condition) was Neurobasal Plus medium (25mM) with B27 supplement Plus, GlutaMAXI, and Antibiotic-Antimycotic. Low glucose media used Neurobasal A Medium (no D-glucose, no sodium pyruvate) supplemented with 5mM glucose, 0.22mM Sodium Pyruvate, B27 supplement Plus, GlutaMAXI, and Antibiotic-Antimycotic (all from ThermoFisher Scientific; Waltham, MA, USA). Cultures were grown in one of the two media conditions throughout the experimental duration. One-half of the media was replaced every two days. Experimental assays were performed at DIV 14 for cortical cultures and DIV 18 for hippocampal cultures. Cortical neuronal cells (DIV 14) from 5 different pregnant mice were grown on 24-well plates in 5mM glucose and 25mM glucose. Cells were lysed and RNA was extracted with a ReliaPrep RNA Miniprep Systems kit (Promega #Z6011) using the manufacturers protocol. The lysates from 8 wells with neurons from the same pup and grown in the same condition (5mM or 25mM) were run through the same column and constituted one sample. Five samples were collected for both the high (25mM) and low (5mM) glucose conditions. We prepared to assess the quality of the RNA with an RNA 6000 Pico kit (Agilent # 5067-1513) using the manufacturers protocol. We then used a 2100 Bioanalyzer instrument (Agilent) to obtain RNA integrity numbers for all samples. RNA extracts were prepared for sequencing with Stranded mRNA Prep (Illumina #20040532) according to the manufacturers protocol. The generated libraries were pooled and sequenced using a NextSeq P3 kit (Illumina #20040560). Sequencing was performed on the NextSeq 2000 platform (Illumina) with 100 paired end reads.