GSM6123034: chr2_Rep1-Rep5 and yfp_Rep1-Rep5; Mus musculus; RNA-Seq
The Dentate Gyrus was dissected from animals that were injected with AAV5-EF1a-DIO-hChR2(H134R)-eYFP and allowed to recover. Animals were then stimulated using the stimulation protocol described previously. After stimulation, animals were transcardially perfused using ice cold PBS solution. The brain was then removed and placed in ice cold PBS and sliced bilaterally across the sagittal midline. Each hemisphere was then taken and the DG was dissected carefully in ice cold PBS. To ensure that mostly stimulated DG tissue was used for sequencing experiments, only the upper half or dorsal DG was then collected. Both Dorsal DG sections from each animal were then placed in a cryogenic tube and flash frozen and placed in -80°C. Single-nucleus RNA sequencing was performed following the SPLiT-seq method with minor modifications (Rosenberg et al., 2018). Nuclei isolated from flash frozen DGs was performed as previously described (Su et al., 2017, Qian et al. 2020). Briefly, tissue was thawed, minced and homogenized using a tissue grinder in a 1 mL of HB buffer (1 mM DTT), 0.15 mM spermine, 0.5 mM spermidine, EDTA-free protease inhibitor, 0.3% IGEPAL-630, 0.25 M sucrose, 25 mM MgCl2, 20 mM Tricine-KOH) for 5 to 10 strokes, then filtered through a 40 mm strainer, under layered with a cushion buffer (0.5 mM MgCl2, 0.5 mM DTT, EDTA-free protease inhibitor, 0.88 M sucrose) to prevent damage to nuclei, and centrifuged at 2800 g for 10 minutes in a swinging bucket centrifuge at 4°C. Pellets of Nuclei were collected. Nuclei were then centrifuged for 3 mins at 500 g at 4°C. The pellet was resuspended in 1 mL of cold PBS-RI (1x PBS + 0.05 U/ml RNase Inhibitor). The nuclei were passed through a 40 mm strainer. 3 mL of cold 1.33% formaldehyde solution was then added to 1 mL of cells. Nuclei were fixed for 10 mins before adding 160 mL of 5% Triton X-100. We then permeabilized nuclei for 3 mins and centrifuged at 500 g for 3 mins at 4°C. Nuclei were resuspended in 500 mL of PBS-RI before adding 500 mL of cold 100 mM Tris-HCl pH 8. Then, nuclei were spun down at 500 g for 3 mins at 4°C and resuspended in 300 mL of cold 0.5 X PBS-RI. Finally, nuclei were again passed through a 40 mm strainer and then counted on a hemocytometer, diluted to 1,000,000 cells/ml. mRNA from single nuclei were tagged 3 rounds with barcoded primers, with in-cell ligations using T4 DNA ligase.   Plates were incubated for 30 mins at 37°C with gentle shaking (50 rpm) to allow hybridization and ligation to occur. The ligation products were purified with Dynabeads MyOne Streptavidin C1 beads. After washing beads once with 10 mM Tris and 0.1% Tween-20 solution and once with water, beads were resuspended into a solution containing 110 mL of 2X Kapa HiFi HotStart Master Mix, 8.8 mL of 10 mM stocks of primers BC_0062 and BC_0108, and 92.4 mL of water. PCR thermocycling was performed as follows: 95°C for 3 mins, then five cycles at 98°C for 20 s, 65°C for 45 s, 72°C for 3 mins. After these five cycles, Dynabeads were removed from PCR solution and EvaGreen dye was added at a 1X concentration. Samples were again placed in a qPCR machine with the following thermocycling conditions: 95°C for 3 mins, cycling at 98°C for 20 s, 65°C for 20 s, and then 72°C for 3 mins, followed by a single 5 mins at 72°C after cycling. Once the qPCR signal began to plateau, reactions were removed.   PCR reactions were purified using a 0.8X ratio of KAPA Pure Beads and cDNA concentration was measured using a qubit. For tagmentation, a Nextera XT Library Prep Kit was used. 600 pg of purified cDNA was diluted in water to a total volume of 5 ml.10 mL of Nextera TD buffer and 5 mL of Amplicon Tagment enzyme were added to bring the total volume to 20 ml. After mixing by pipetting, the solution was incubated at 55°C for 5 mins. A volume of 5 mL of neutralization buffer was added and the solution was mixed before incubation at room temperature for another 5 mins. PCR was then performed with the following cycling conditions: 95°C for 30 s, followed by 12 cycles of 95°C for 10 s, 55°C for 30 s, 72°C for 30 s, and 72°C for 5 mins after the 12 cycles. 40 mL of this PCR reaction was removed and purified with a 0.7X ratio of SPRI beads to generate an Illumina-compatible sequencing library. SPLiT-seq