GSM2609372: 4_1hr_F_CA1; Mus musculus; RNA-Seq
The nuclei dissociation protocol is similar to the previously described protocol with some modifications. Hippocampus was carefully excised and immediately placed into a nuclei isolation medium (sucrose 0.25 M, KCl 25 mM, MgCl2 5 mM, TrisCl 10 mM, dithiothreitol, 0.1 % Triton, protease inhibitors). Tissue was Dounce homogenized, allowing for mechanical separation of nuclei from cells. The nucleic acid stain Hoechst 33342 (5 µM, Life Technologies) was included in the media to facilitate visualization of the nuclei for quantification, but excluded for sorting. Samples were washed, resuspended in nuclei storage buffer (sucrose, MgCl2 5 mM, and TrisCl 10 mM) and filtered. Solutions and samples were kept cold throughout the protocol. For RNA-seq experiments, tools and solutions were made RNAse-free and RNAse inhibitors were used. Library preparation followed the Smart-seq2 protocol. The Nextera XT kit was used for sample preparation following all transcriptome amplifications. Single-nuclei and amplified bulk samples were measured by Picogreen (LifeTech) and normalized so that samples were at a concentration of 0.3 ng/µl. Samples were first subjected to a tagmentation reaction, indexed, and PCR amplified. Libraries were then mixed in 48-sample pools and purified with magnetic beads (AMPure). Quality control checks for the library preparations included electrophoresis (Agilent Technologies 2100 Bioanalyzer). Samples were sequenced in the Salk Institute Next Generation Sequencing Core on an Illumina HiSeq 2500 high-throughput sequencing system with single-end 50bp reads.