title: GSM3504095: LS-15362_S25_E1-50; Mus musculus; RNA-Seq
library_construction_protocol: Tissue samples were obtained from adult male and female transgenic mice carrying a Cre transgene and a Cre-reporter transgene. After euthanasia, the brain was rapidly dissected and mounted for coronal slice preparation on a Compresstome VF-300 vibrating microtome (Precisionary Instruments) and sectioned at  250 μm intervals.     For whole cell dissociation, 2 mg/ml of pronase in ACSF solution was added to dissected tissues and enzymatic digestion was carried out at room temperature. After digestion,The tissue was triturated and the cell suspension was incubated on ice in preparation for fluorescence-activated cell sorting (FACS). Live cells were sorted by excluding DAPI positive events and debris, and gating to include red fluorescent events (tdTomato-positive cells). Single cells were collected into strip tubes containing 11.5μl of collection buffer (SMART-Seq v4 lysis buffer), RNase Inhibitor, and ERCCs (External RNA Controls Consortium, MIX1 at a final dilution of 1x10-8) and tubes were then stored at -80°C.    For nuclei isolation, dissected tissues were placed into a homogenization buffer (10mM Tris pH 8.0, 250mM sucrose, 25mM KCl, 5mM MgCl2, 0.1% Triton-X 100, 0.5% RNase inhibitor, 1X protease inhibitor, and 0.1mM DTT). Tissues were placed into a 1ml dounce homogenizer and homogenized using 10 strokes of the loose dounce pestle followed by 10 strokes of the tight pestle to liberate nuclei . Mouse anti-NeuN antibody (EMD Millipore, MAB377, Clone A60) was added to the nuclei at a final dilution of 1:1000 and nuclei suspensions were incubated at 4°C for 30 minutes, followed by incubation in secondary antibody (goat anti-mouse IgG (H+L), Alexa Fluor 594 conjugated, ThermoFisher Scientific, 1:5000) for 30 minutes at 4°C and staining with DAPI. Single nuclei were captured by gating on DAPI-positive events, excluding debris and doublets, and then gating on Alexa Fluor 594 (NeuN) signal. Strip tubes containing FACS isolated single nuclei were then frozen at -80°C. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech #634894) was used per the manufacturer's instructions for reverse transcription of single cell RNA and subsequent cDNA synthesis. Mouse whole cells were subjected to 18 PCR cycles after the reverse transcription step, whereas mouse nuclei were subjected to 21 PCR cycles.     Sequencing libraries were prepared using NexteraXT (Illumina, FC-131-1096) with NexteraXT Index Kit V2 Set A (FC-131-2001). NexteraXT libraries were prepared at 0.5x volume, but otherwise followed the manufacturer's instructions.
attributes.tag: GEO Accession
attributes.value: GSM3504095