GSM4628212: E3Ki_E4Mixed (scRNA-seq); Homo sapiens; Mus musculus; RNA-Seq
Hippocampi were isolated from single hemispheres of mice at the time of euthanasia, immediately frozen in dry ice and stored at -80°C. For isolation of human nuclei, hippocampi were thawed and incubated in HEB (BrainBits LLC)(1mL/ hippocampi) and kept on ice. 2mL chilled lysis buffer was then added to the tissue and was homogenized with 21G needle. Tissue was lysed on ice for 15 minutes and swirled to mix. This was repeated 3 times during incubation. Lysed hippocampi were titrated 5-7 times with 1mL pipette. Lysate was then washed in 30mm MACS Smart Strainer to remove large clumps and then centrifuged at 500g for 5 minutes are 4°C. Supernatant was aspirated and pellet was washed with 1mL Nuclei Wash and Resuspension Buffer. Nuclei were centrifuged at 500g for 5 min at 4°C and supernatant was then aspirated. The nuclear pellet was resuspended in 500mL staining buffer (0.5% RNase-free BSA and 0.2 mg/mL RNasin Plus RNAse inhibitory in RNAse-free PBS) and incubated for 15 minutes on ice to allow for blocking of non-specific antibody binding. 100mL of samples were removed for negative control staining. After blocking, anti-human nuclear antigen antibody conjugated to phycoerythrin was added to the nuclei solution [1:100] and incubated at 4°C for 30 minutes rotating. After incubation, samples were washed by adding 500mL of staining buffer to each tube and inverting several times. Samples were then collected by centrifugation for 5 min at 400g at 4°C. Supernatant was aspirated, and nuclei were resuspended in 500mL staining buffer for FACS sorting, with DAPI (0.1 mg/mL) added directly before sorting on Aria FACS sorter (BD Biosciences). cDNA libraries were prepared using the Chromium Single Cell 3′ Library and Gel Bead kit v3 (10x Genomics: 1000092) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer at the UCSF CAT Core.
GEO Accession
GSM4628212