title: GSM4151314: H1; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with artificial cerebrospinal fluid under anesthesia state which may cool the brain more rapidly(Zeisel et al., 2018). We slightly modified their protocol. ACSF consisting of 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 75 mM sucrose, 20 mM glucose, 1 mM CaCl2, 7 mM MgSO4 and 0.1 µM tetrodotoxin was equilibrated in 95% O2 /5% CO2 before use. Mice were anesthetized with 1% pentobarbital sodium, followed by transcardial perfusion through the left ventricle with ice-cold ACSF. The brain was removed, and 300-μm vibratome sections were collected using Vibroslice (VT 1000S; Leica) in ice-cold ACSF.  The hippocampus was microdissected and transferred to 100-mm dishes containing 7 mL digestion solution (2 mg/mL papain, Worthington, LS003119; 0.5 mM EDTA-2Na; 2.5 mL/L Glutamax 100X, ThermoFisher; resolved in Hibernate A, ThermoFisher) with 30-40 min enzymatic digestion at 30 ℃. The digestion solution was weakly acidic after the addition of papain, and 300-μL neruobasal A (ThermoFisher, 10888022) was required to adjust the pH to 7.4. After incubation, the tissue was transferred to a 15-mL tube containing 5 mL ice-cold HABG (60 mL Hibernate A; 20 mL/L B27 50X, ThermoFisher; 2.5 mL/L Glutamax 100X). The tissue pieces were gently triturated through Pasteur pipettes with polished tip openings of 0.6 mm, 0.3 mm and 0.15 mm diameter. The supernatant was transferred to a 15-mL tube after settling for 1 min. The sediment was resuspended in 2 mL HABG, and the supernatant was transferred. The procedures were repeated until the sediment was almost dissociated. The cells were enriched by centrifugation (800 g, 5 min, 4 ℃), resuspended in 1 mL HABG containing 1 mg/mL DNase I (Roche, 11284932001), and filtered with a 40-μm cell strainer (Falcon). To exclude dead cells, 0.1 μg/mL DAPI (Invitrogen) was added to the single-cell suspension. Then, the cells were immediately loaded into the Beckman MoFlo XDP Cell Sorter system for fluorescence-activated cell sorting (FACS). The high mCherry and low DAPI fluorescent cells were isolated into 1.5-mL tubes containing 5.4 µL sample buffer, 0.6 µL RNase inhibitor and 4 µL RNase-free water (Discover-sc WTA Kit V2, N711, Vazyme). Oxygenation and a short time of dissection were crucial to keep a high rate of survival in the cell suspension. For cell sorting experiments, ∼500 - 1,000 cells were isolated from 2 - 3 mice, and this represented 1 biological sample in our analysis. For each group, 3-5 biological replicates were obtained. RNA libraries were prepared for sequencing using standard Illumina protocols
attributes.tag: GEO Accession
attributes.value: GSM4151314