GSM7597946: Mouse #784, APP/PSEN1, synaptosome; Mus musculus; ssRNA-seq
First, synaptosomes were fractionated from frontal cortex brain tissue according to the Whittaker et al. (1964) protocol. Approximately 300 mg of brain tissue was homogenized using a glass douncer in 1.5 mL of homogenization buffer: 0.32 M sucrose, 1 mM sodium bicarbonate, 1 mM magnesium chloride, 0.5 mM calcium chloride, 100 μM aurintricarboxylic acid, 24 U/mL NEB Murine RNAse inhibitor, and 0.5x Thermo HALT protease and phosphatase inhibitor cocktail. Following homogenization, 120 μl of homogenate was set aside for RNA extraction and sequencing and additional analyses. The remaining homogenate was spun down at 750 × g for 10 min at 4ºC to remove debris. Next the supernatant was centrifuged at 13,800 × g to produce a pellet of crude synaptosomes. The supernatant was collected as the cytoplasmic fraction. The crude synaptosome pellet was resuspended in 1 mL of homogenization buffer. A discontinuous sucrose gradient was prepared in a 4.2 mL ultracentrifuge tube with a 1 mL bottom layer of 1.2 M sucrose + 1 mM sodium bicarbonate, 1 mL middle layer of 1.0 M sucrose + 1 mM sodium bicarbonate, and a 1 mL top layer of 0.85 M sucrose + 1 mM sodium bicarbonate. The 1 mL of resuspended crude synaptosome was layered on top of the gradient. The samples were then spun using a swinging bucket rotor in an ultracentrifuge at 28,000 rpm (~82,000 × g). The purified synaptosomes were collected from the interface between the 1.2 M and 1.0 M layers. RNA was subsequently extracted from 500 μl of the collected synaptosomes with Trizol/chloroform. We used the total stranded RNA with ribo-removal kit and followed the corresponding protocol from Illumina, product: 20040529; rna libraries from each sample (mouse, homogenate or synaptosome) were individually barcoded and pooled in batches of 12 (6 homogenate + 6 synaptosome)