GSM6199123: P10 Area Postrema/NTS region; Mus musculus; RNA-Seq
To identify the area postrema (AP) during microdissection, 50 µl of a 1% Evans Blue solution was administered transcardially to a male P10 mouse previously anesthetized with a single i.p injection of Avertin (250 mg/kg – Sigma-Aldrich #T48402). After 5 min, the mouse was decapitated, and the brain was dissected and placed into a petri dish with ice cold sterile 1X PBS under a dissecting microscope. After visually identifying the AP (in blue), the AP-centric region was microdissected and immediately dissociated into a single cell suspension. The dissociation was carried out according to Saxena et al (40) with volumes adjusted for tissue size. Briefly, the tissue was triturated and incubated with Papain-DNase1 solution using the Worthington Papain Dissociation System (#LK003150, Worthington, NJ) for 60 minutes at 37C with constant agitation. All components of the dissociation system were equilibrated with 95% O2: 5% CO2 before use. The tissue was then further triturated by pipetting until a homogenous cell suspension was observed. The suspension was then centrifugated and resuspended in 0.04% BSA Library preparation was performed by the UCSF Genomics Core according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics)