title: GSM7503686: KO3, snRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Hippocampi were isolated and, for each animal, a punch was collected from the middle of the hippocampus using a 1 mm diameter tissue punch. The punches were immediately stored at -80oC. For each animal, tissue punches from the right and left hippocampi were combined and homogenized using a glass Dounce homogenizer in 500 ml ice-cold Nuclei EZ lysis buffer (NLB) (NUC-101, Sigma) 25 times with pestle A and then 25 times with pestle B. The mixture was transferred to a pre-chilled Eppendorf tube and 1000 ml of NLB was added to the tube and incubated on ice for 5 minutes (min). The tubes were centrifuged at 800 x g for 5 min at 4o C. The nuclei were resuspended in 75 ml of nuclei suspension buffer (consisting of 1X PBS, 1% BSA (AM2618, Thermo Fisher Scientific), and 0.2 U/ml (1%) Rnase inhibitor (AM2694, Thermo Fisher Scientific)). The nuclei were then filtered through a 40 mm Flowmi Cell Strainer (H13680-0040, Bel-Art) to remove cellular debris. Concentration of nuclei for each sample was calculated using Trypan Blue (T8154, Sigma) on a hemocytometer, aiming for a final concentration of ~1000-2000 nuclei/ml. Single nuclei were processed using the 10X Genomics Next GEM Single Cell 3' Reagent Kit v3.1 following the manufacturer's user guide. All mRNA samples were examined for quantity and quality by NanoDrop and Bioanalyzer 2100 (Agilent). The libraries were constructed following TruSeq Stranded mRNA Sample preparation guide (Illumina) and paired end sequencing was performed on the Illumina NovaSeq 6000 platform at the McDermott Center Next Generation Sequencing Core at UTSW.