title: GSM9117426: PVH-neuron nucleus, retrograde HSV, sample 169, lib. 1; Mus musculus; RNA-Seq
library_construction_protocol: C57BL/6J or H2B-TRAP mice aged 6-12 weeks recieved bilateral stereotaxic injections of AAVDJ-hSyn-DIO-H2B-mCherry and/or AAVrg-hSyn-Cre, respectively. After 2-3 weeks of recovery, mice were sacrificed between 9 am – 12 pm by rapid decapitation immediately after removal from the home cage. Brains were extracted and chilled in DMEM/F12 media slush. Next, brains were placed ventral side up in a chilled stainless steel brain matrix (Roboz Surgical Instrument Co.: SA-2165) and 1 mm coronal sections of the hypothalamus were collected and treated with RNAlater (QIAGEN catalog #76106). The PVH was then microdissected under a fluorescent stereoscope and tissue was pooled and stored at -80. Each sample underwent nuclear dissociation and purification procedures and were incubated with NucBlue (Thermofisher scientific). After incubation, nuclei were flow sorted together into 96 well plates with each well containing sNuc-seq lysis buffer (Todd et al., 2020; Tao, et al., 2021) using a Beckman Coulter MoFlo Astrios EQ with a 70 μm nozzle. Single nucleus samples were processed into cDNA libraries, and sequenced by Next-Seq 500 (SR75, high-output kit) as previously described (Todd et al., 2020). Reads were demultiplexed by bcl2fastq2 v2.20.0 (Illumina) and aligned to the mouse genome by STAR v2.6.1 (Dobin et al., 2013). Duplicates were removed with Picard Tools v2.18.21. Aligned reads were processed into a digital gene expression (DGE) file with Drop-Seq Tools v2.3.0 and tagged using “GENCODE_M16_PRI” annotation. The DGE file was input to the single-cell analysis R package, Seurat (Stuart et al., 2019), version 5, for further analysis, using default settings except where noted.