title: GSM7116172: POA-US; Mus musculus; RNA-Seq
library_construction_protocol: Approximately 1 h after ultrasound stimulation, mice were transcardially perfused with ice-cold phosphate-buffered saline (1X PBS). The POA was extracted from each brain and homogenized in a Dounce Homogenizer (Kimble) containing 0.4 mL of freshly prepared nuclear extraction buffer [250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris-HCl (pH8.0), 0.1% Triton X-100,  0.2U/μl recombinant ribonuclease inhibitor  (Invitrogen™ RNaseOUT™)] applying 4 strokes with the A pestle followed by 8 strokes with the B pestle. The homogenate of four mice from the control group and four mice from the US group was pooled for nucleus isolation. Samples were filtered through a 30 µm cell strainer tube (Corning™ Falcon™) and centrifuged at 500xg for 5 min at 4°C to precipitate nuclei. The dissociated nuclei were washed once with and gently resuspended with nuclear storage buffer (PBS with 1% BSA, 0.2U/μl recombinant ribonuclease inhibitor). To purify neuron nuclei for single-nucleus RNA-seq, the nuclei suspension was incubated with Alexa Fluor®488 conjugated mouse anti-NeuN antibody (Millipore sigma, MAB377X, 1:200) for 30mins on ice. After incubation, the nuclei were washed once with and resuspended with nuclear storage buffer with 1ug/ml DAPI. Samples were filtered through a 30 µm cell strainer tube and shortly sorted using FACS (MoFlo, Beckman Coulter) using FlowJo (v10.8) software. The neuron nuclei were sorted on DAPI+/NeuN+ nuclei and collected into 1.5 ml Eppendorf tubes. Approximately 25,000 neuron nuclei were collected from each of the control and US groups and sent to GTAC@MGI for single-cell RNA-seq (10X Genomics). Using a Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics), neuron nuclei were immediately loaded onto a Chromium Single Cell Processor (10X Genomics) for RNA barcoding from single nuclei. Sequencing libraries were constructed according to the manufacturer's instructions, and resulting cDNA samples were run on an Agilent Bioanalyzer using the High Sensitivity DNA Chip to determine cDNA concentrations. The samples were combined and run on an Illumina Nova Seq 6000.