GSM7844863: PVH, hM3D expressed, VPA exposed and PND2 CNO treated, snRNA-seq, rep1; Mus musculus; RNA-Seq
Male OT-Cre; Ai9 mice at 5 weeks of age were deeply anesthetized by isoflurane (Fujifilm, #099-06571), perfused by cold phosphate-buffered saline (PBS) to remove blood cells, and euthanized by decapitation. Brains were sectioned into 1000-μm coronal slices under microscopy and the sections were floated in 1% BSA-PBS (Nacalai Tesque, #0128197 and Takara, #T9181). The PVH was dissected based on the fluorescence of tdTomato in OT-Cre; Ai9 male mice, and homogenized sufficiently in ice-cold Nuclei EZ Lysis Buffer (Millipore Sigma, N-3408). The resulting suspension was incubated on ice for 5 min and filtered through a 70-μm strainer. The nuclei were pelletized by centrifugation at 500xg and 4 ℃ for 5 min, and the supernatant was removed. The nuclei were resuspended in Nuclei EZ Lysis Buffer and pelletized again by centrifugation at 500xg and 4 ℃ for 5 min. They were then resuspended in 1% BSA-PBS containing 0.2 U/μL RNase Inhibitor (Roche, 3335399001) (Resuspension Buffer). We utilized 2–4 male mice for each condition, pooled the isolated nuclei, and centrifuged again at 500xg and 4 ℃ for 5 min to pellet the nuclei. The nuclei were then resuspended in resuspension buffer and 10 μg/mL DAPI, and filtered through a 40-μm strainer. The nuclei were sorted from the suspension using a cell sorter (SH800Z; Sony) at 5 ℃. The gate was set first to identify a single nucleus population based on the DAPI signal and then to select larger nuclei preferentially, inferring neurons while simultaneously excluding multiplets. Immediately after sorting was completed, the nuclei stained with DAPI were counted using a hemocytometer to verify the yield of intact nuclei.  For the recovery experiments, we utilized male OT-Cre; stop-hM3 mice at 5–7 weeks of age that had been exposed to VPA prenatally and CNO or saline as a control at PND 2. We followed the above protocols with slight modifications. Briefly, OT-Cre; stop-hM3 mice were injected with AAV in the PVH and the supraoptic nucleus regions at three weeks of age to facilitate visualization of OT neurons during the dissection process. To optimize the yield in collecting neural cell nuclei, the FACS gating was adjusted to accommodate larger nuclei. Utilizing the 10X Genomics Chromium platform, we crafted single-nucleus RNA libraries targeting a total of 8,000 nuclei per condition for OT-Cre; Ai9 batches. The library preparation was accomplished using the 10X Genomics RNA 3' v3.1 kit (#1000269). Subsequently, the completed libraries were subjected to sequencing to a depth of 110 gigabases using the HiSeqX platform (performed by Novogene, Inc.). For OT-Cre; stop-hM3 mice batches, the aim was to isolate 5,000 nuclei using the Chromium system.