GSM6603752: mature, 4hr, X100119_130; Mus musculus; RNA-Seq
The hippocampus was dissected bilaterally after deep anesthesia with ketamine/xylazine/acepromazine and cervical dislocation. Hippocampal tissue was immediately placed into a nuclei isolation medium (sucrose 0.25 M, KCl 25 mM, MgCl2 5 mM, TrisCl 10 mM, dithiothreitol 100 uM, 0.1% Triton, protease inhibitors). Tissue was dounce homogenized and then samples were washed, resuspended in nuclei storage buffer (0.167 M sucrose, MgCl2 5 mM, and TrisCl 10 mM, dithiothreitol 100 uM, protease inhibitors) and filtered. Antibody staining proceeded in the nuclei storage buffer. Solutions and samples were kept cold throughout the protocol. Tools and solutions were made RNAse-free and RNAse inhibitors were used (Ambion #AM2684 at 1:1000 in both isolation and storage buffers). Samples were run on a BD Influx cell sorter and collected directly into 384-well plates pre-filled with 1 uL lysis buffer (0.1% Triton-X, 1 U/ul RNAse inhibitor, 2.5 mM dNTP mix, 2.5 uM anchored oligo-dT primer) and frozen at -80C until further processing Libraries were prepared using the Smart-seq2 protocol with minor modifications. First strand synthesis with ProtoScript II reverse transcriptase (New England Biolabs), PCR preamplification with KAPA Hifi HotStart ReadyMix (Kapa), and bead cleanup (Agencourt AMPure XP beads, Beckman Coulter) were performed with the aid of a Mosquito HV liquid handler (SPT Labtech). The quality of cDNA was assessed by Bioanalyzer chip (Agilent) and concentration quantified by Qubit assay (Invitrogen). Single nuclei libraries were prepared with a Nextera XT DNA library prep kit (Illumina) and pooled together for sequencing. The quality of pooled libraries was assessed by TapeStation assay (Agilent). Libraries were sequenced on an Illumina HiSeq using single-end 50bp reads by the Salk Institute Next Generation Sequencing Core.