GSM6602012: EC.APP.1; Mus musculus; RNA-Seq
Mice were anaesthetized with isoflurane before decapitation. Brains were dissected on ice and rested in pre-bubbled slurry cutting solution [250 mM sucrose, 26 mM NaHCO3, 10 mM [D+] glucose, 3 mM myo-inositol, 2.5 mM KCl, 2 mM sodium pyruvate, 1.25 mM NaH2PO4·2H2O, 0.5 mM ascorbic acid, 1 mM kynurenic acid, 0.1 mM CaCl2 and 4 mM MgCl2] for 1 min. A downward cut across the midbrain was made to separate the cerebellum from the frontal cortex. To dissect DCN, the cerebellum was first sectioned at 300µm in cold cutting solution using a vibratome [Leica VT-1200]. Approximately 4-5 sections were used to microdissect the DCN in chilled pre-bubbled artificial cerebrospinal fluid [ACSF: 10 mM [D+] glucose, 126 mM NaCl, 24 mM NaHCO3, 1 mM NaH2PO4·2H2O, 2.5mM KCl, 0.4mM ascorbic acid, 2 mM CaCl2 and 2 mM MgCl2] under a light microscope. For EC dissection, a longitudinal cut was made to separate the two hemispheres. With the medial surface of the hemispheres facing up, the thalamic and hypothalamic region were scooped outpeeled back and removed, exposing the hippocampal formation. The hippocampus region was further rolled out, with the hippocampus remaining attached to the EC via the subiculum. The entorhinal cortex was then dissected by making four cuts: one vertical cut was made parallel to the join between the hpc and EC, roughly one hippocampus width away; then horizontal cuts were made at the top and bottom of the join, and finally a vertical cut was made to sever the EC and hippocampus. Dissected tissues were transferred to chilled homogenisation buffer [0.25 M sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tricine, pH 7.8] in a Dounce homogeniser and homogenised with 10 strokes of the pestle. To lyse the samples, NP-40 detergent was added to obtain a final concentration of 0.3% and samples were dounced with 5 additional strokes. Lysates were filtered using 40um FlowMi tip strainers to remove debris and clumped nuclei. Each 2ml sample was gently mixed with 4.6ml 1.8M sucrose cushion buffer [1.8M sucrose, 10mM Tris-HCl, 1.5 mM MgCl2, pH 6.9] and layered over a 3ml sucrose cushion. Samples were spun in an ultracentrifuge at 30,000 x g for 45 mins at 4°C. The supernatant was then removed and pellets were resuspended in 90ul nuclei resuspension buffer [10mM Tris-HCl, 0.25 M Sucrose, 25 mM KCl, 1.5 mM MgCl2, 1.5 mM CaCl2, pH 6.9]. Nuclei were inspected and counted under a microscope using a hemocytometer and Trypan blue dye. cDNA libraries were generated according to manufacturer's protocol (10x Genomics, Inc.). In brief, approximately 10, 000 nuclei per sample were processed using the Chromiium Single Cell 3' v3 Gene Expression kit. The libraries were subsequently sequenced using NovaSeq6000 (NovogeneAIT Genomics).