GSM9149736: PFC CC025 Pooled Sample #1; Mus musculus; RNA-Seq
Briefly, mice were sacrificed with an overdose of isoflurane, followed by transcardial perfusion with carbonated (95% O2, 5% CO2) Hanks' Balanced Salt Solution (HBSS) containing small-molecule cocktail consisting of 1 μM TTX (Sigma), 100 μM AP-V (Thermo Fisher Scientific), 5 μg actinomycin D (Sigma) per milliliter and 10 μM triptolide (Sigma) to optimize for preserving transcriptional state. Brains were removed, 500μm sections were collected, and the region of interest was dissected (ANT or ACC). The tissue was dissociated using papain (Worthington, cat. no. LS003124) dissolved in Hibernate A buffer (Fisher Scientific, cat. no. NC1787837) containing 1 μM TTX, 100 μM AP-V (Thermo Fisher Scientific), 5 μg actinomycin D (Sigma) per milliliter, 10 μM triptolide (Sigma), and 10 μg anisomycin (Sigma) per milliliter, and incubated for 25-30 min at 37C, followed by manual trituration using fire polished Pasteur pipettes and filtering through a 40μm cell strainer (Millipore Sigma, cat. no. BAH136800040). Cells were washed with a wash buffer (PBS + 1% BSA) and centrifuged at 500 g for 5 min, the supernatant was carefully removed, and cells were resuspended in ~500ul wash buffer and 10% DAPI. Single cell RNA- sequencing was carried out with the Chromium Next GEM Single Cell 3' Kit v3.1 (10X Genomics, cat. no. 1000268). Manufacturer's instructions were followed for downstream cDNA synthesis (12-14 PCR cycles) and library preparation.