GSM8253395: v3.9.4; Callithrix jacchus; RNA-Seq
Tissue dissection for cryosection and nuclei isolation: Before the day of tissue harvest, a custom-made brain holder was generated for each marmoset brain by 3D printing (Ultimaker 2+) to guide tissue sampling (141). Tissue dissection was carried out as described (34). Briefly, marmosets were deeply anesthetized with 5% isoflurane until no signs of breathing, then were transcardially perfused with ice-cold artificial cerebrospinal fluid (aCSF) for 5 min. After skull and meninges removal, each brain was quickly positioned into the designated brain holder submerged in ice-cold aCSF solution within 10 min post-perfusion. The brain was sectioned into 12–13 slabs at 3 mm with a homemade blade-separator set submerged in aCSF solution. Each brain slab was transferred into a tissue cassette (70078-15, Electron Microscopy Sciences) with the anterior side of the brain slab facing the biopsy foam pad (62325-06, Electron Microscopy Sciences). The cassettes were then submerged into a jar full of RNAlater (RNAlater™ Stabilization Solution, AM7021, Invitrogen) and stored at 4°C overnight. The following day, brain slabs were transferred to 25 x 20 x 5 mm3 molds (Tissue-Tek® Cryomold®, 4557, Sakura Finetek) on ice to facilitate targeted sampling. Slabs were matched to terminal MRI and informed by marmoset 3D MRI atlases (139,140) for region annotation. For nuclei dissociation, a cylinder of tissue 2 mm in diameter and 3 mm in height for each area was collected with a tissue punch (EMS-core sampling tool, 69039-20, EMS), and ejected into PCR tubes filled with 100 µL of RNAlater and kept at −80°C for long-term storage. For cryosection, a 6.5 x 6.5 x 3 mm3 block of tissue per region of interest (ROI) was prepared by a customized 3D-printed brain cutter and stored in cryomold filled with RNAlater at −80°C or proceeded immediately to OCT (Tissue-Tek® O.C.T. Compound, 4583, Sakura) embedding. The quality of RNAlater-preserved tissue was assessed by measuring RNA Integrity Number (RIN) on the Agilent 2100 Bioanalyzer (G2939BA, Agilent). Bulk RNA was isolated with TRIzol™ Reagent (15596026, Invitrogen) and measured with Agilent RNA 6000 Pico Kit (5067-1513, Agilent); samples with RIN >8.5 were used in the study. 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit v3.1