title: GSM7964435: ds2, 4w, right, snRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Mice were deeply anesthetized (ketamine/xylazine, as described above), brains were carefully removed and placed into ice cold Earl's balanced salt solution (EBSS, in mM: 117 NaCl, 5.4 KCl, 1 NaH2PO4, 26 NaHCO3, 5.6 glucose, 1.8 CaCl2 .2H2O and 0.8 MgSO4) with trehalose 5% v/v and kynurenic acid 0.8 mM. Dissection solution was equilibrated in 95% O2/5% CO2 before use. The brain was cut along the longitudinal fissure and the regions posterior to lambda were cut off. Under a dissection microscope and upon removal of the diencephalon, the medial side of the hippocampus was exposed. The dentate gyrus was isolated by inserting a sharp-needle tip and sliding it superficially along the septotemporal axis of the hippocampus. The dissected tissue was placed in a 0.5 ml microcentrifuge tube with a minimal amount of media, flash-freezed on dry ice and stored at -80ºC until use. Nuclei were isolated as previously described with several modifications. Dounce tissue grinder and pestles were sequentially washed with 100% EtOH, RNAse Zap (Sigma, Cat # R2020), 2-3 rounds with RNAse-free water, and finally rinsed with EZ Lysis Buffer (Sigma, Cat # NUC-101). All material and buffers were chilled on ice. The tissue was transferred to a chilled dounce prefilled with 2 ml ice-cold EZ Lysis Buffer and homogenized slowly with 20-25 strokes of pestle A, followed by 20 strokes with pestle B. Suspension was transferred to a chilled tube and incubated on ice 5 min. Then the suspension was centrifuged at 500 × g for 5 min at 4°C and the supernatant was removed. The pellet was resuspended in 4 ml ice-cold EZ Lysis Buffer and after a second round of ice incubation and centrifugation, the isolated nuclei were resuspended in 4 ml ice-cold Nuclei Suspension Buffer (NSB) (1x RNase-free molecular-biology grade PBS, 0.1% molecular-biology grade BSA 100 μg/ml and 0.2U/μl RNase inhibitor). Finally, nuclei were centrifuged at 500 × g for 5 min at 4°C and after removing the supernatant were resuspended in 1ml NSB-Ruby (final Ruby concentration 1:500) and filtered twice through a 35-μm cell strainer to remove as many debris as possible. Nuclei were kept on ice until sorting (Harvard University, Bauer Core Facility, BD FACSAria II) into 96 well plates, pre-coated with BSA to reduce adherence and improve recovery, containing with ~10-20 μl of rich-NSB (with final 2U/μl of RNase inhibitor and 1% BSA). The final nuclei concentration was determined using a C-chip Neubauer counting chamber and Trypan Blue (1:2 final dilution). Nuclei were immediately loaded for single-cell GEM formation (10x Genomics, single cell RNA sequencing 3ʹ, Chromium v3.1) Dataset sampling for snRNA-seq experiments was carried out within a minimal time interval, processing two time points in a single experiment each day to minimize batch effects. For each time point, four dentate gyri (left or right hemispheres) from male and female mice were pooled. For snRNA-seq, nuclei were loaded in the 10x Genomics chips aiming to recover 2,000–10,000 nuclei. cDNA amplification and library construction were done following 10x Genomics protocols. Libraries were generated using Chromium v3.1, quantified in BioAnalyzer and sequenced on an Illumina HiSeq 2500. Samples were sequenced to a depth of 40–70,000 reads per cell. snRNA-Seq