GSM3737075: SUS3; Mus musculus; RNA-Seq
On the third day from the end of the last PR session, mice were deeply anesthetized with a ketamine/xylazine mixture and cardially perfused with ice-cold PBS. Mice were then decapitated and brains were extracted. Two slices (1.0 mm thick) containing the NAc were made using an ice-cold brain matrix (ZIVIC instruments). Bilateral 12-gauge punches containing NAc were obtained, pooled from 2–3 mice per condition (susceptible, resilient, or drug-naïve), and kept in ice-cold dissociation media [Hibernate-A media (BrainBits) containing 5 μM DL-APV, 12.5 unit/ml DNase I, and 5% D-(+)-Trehalose dehydrate] until the dissociation procedure was initiated. A pure population of cholinergic interneurons was obtained via fluorescence-activated cell sorting (FACS) according to a previously published protocol, with minor modifications. Briefly, the punches were dissected out using fine forceps, chopped with scalpels, transferred into dissociation medium containing papain enzyme (1 mg/ml, Worthington), and dissociated for 45 min at 37ºC to yield single cells. Dissociated cells were triturated and filtered through a 70-µm mesh into 50 ml tubes on ice. To remove excess debris and inactivate residual papain activity, the cell suspension was subjected to centrifugation on an AOI discontinuous gradient [Hibernate-A media (BrainBits) containing 5 μM DL-APV (Sigma), 12.5 unit/ml DNase I, 5% D-(+)-Trehalose dehydrate (Sigma), and albumin ovomucoid inhibitor (AOI, 0.6 mg/ml; Worthington)] at 900 rpm for 6 min at 4ºC. The cell pellets were suspended in dissociation medium and centrifuged at 1250 rpm for 6 min at 4ºC. Cell pellets were re-suspended in ice-cold PBS and immunolabeled with Alexa Fluor 488-anti-ChAT (1:50, abcam) at 4°C for 30 min. Alexa Fluor 488-immunolabeled cells were sorted on a MoFlo® Astrios EQ high speed cell sorter (Beckman Coulter) for GFP signals (detecting Alexa Fluor 488). Cells (5–50 × 10e3 per sample) were collected using FACS. Immediately after sorting, collection tubes were vortexed and flash frozen on dry ice. Samples were stored frozen in Trizol LS at −80°C until RNA extraction and library preparation. RNA was isolated using a Directzol RNA-Mini prep (Zymo). Samples were incubated for 15 min with DNase according to the manufacturer's instructions. RNA was eluted into 12 µl H2O to ensure a viable concentration of RNA for an 8 µl library prep input volume limit. Libraries were prepared using SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Clontech), and validated using an Agilent Bioanalyzer (Agilent TapeStation) and PCR prior to sequencing.
GEO Accession
GSM3737075