GSM8174621: VP mouse, control diet, 2 weeks; Mus musculus; RNA-Seq
For experiments utilizing mice, all mice were anesthetized with isoflurane and transcardially perfused with ice-cold NMDG buffer. Brains were extracted and sliced on a fresh-tissue vibratome. Typically, 4 tissue slices of 300µM thickness were collected for bilateral VP mi-crodissection with a scalpel, resulting in 8 VP tissue samples. Samples were flash-frozen on dry ice and transferred to -80 C for storage. For experiments utilizing rats, rats were injected with 0.7 mL Euthasol for terminal anesthesia, and then perfused with ice-cold NMDG buffer. VP tissue samples were collected using the same procedure employed for mice excepting for with 400µM slices. We also collected 8 NAc tissue samples from the same rats. VP tissue samples from each experimental group (mice) and from all rats were pooled together for a total of 5 nuclear isolation batches by combining samples in a glass Dounce homogenizer containing lysis buffer. We did the same for NAc tissue samples from rats for an additional nuclear isolation batch. After homogenization and a five-minute incubation, samples were diluted using wash buffer, passed through a 40 uM cell strainer, and centrifuged at 500 x g for 10 minutes at 4 C to remove debris and pellet nuclei. This pellet was resuspended in a homogenate solution and combined 1:1 with a 50% iodixanol solution. To further clean up the nuclei pellet using density gradient centrifugation, we underlaid the resulting 25% iodixanol layer with a 30% layer and centrifuged at 3000 x g for 15 minutes at 4 C. We used the 10X Genomics Chromium 3' Next GEM kit v3.1 (Dual Index) to generate cDNA libraries from each nuclear isolation batch. Isolated nuclei preparations were diluted to a concentration of 1,000/uL and loaded onto the 10X chip, targeting capture of 10,000 single barcoded nuclei. We loaded rat nuclei preparations into two lanes on the chip, resulting in two VP and two NAc reactions. We then prepared libraries for sequencing following the Next GEM protocol.