title: GSM9117450: multiplexed sample set 1, PVH nuclei, GEX; Mus musculus; RNA-Seq
library_construction_protocol: Sim1-Cre and H2B-TRAP mice aged 6-12 weeks recieved bilateral stereotaxic injections of AAVDJ-hSyn-DIO-H2B-mCherry 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. The PVH was then microdissected under a fluorescent stereoscope and tissue was pooled by experiment (Sim1-Cre or projection-specific labeling). Each sample underwent nuclear dissociation and purification procedures and were incubated with NucBlue (Thermofisher scientific) as well as an individual hashtag antibody (BioLegend). After incubation, nuclei were flow sorted together into RT Reagent B (10x Genomics) using a Beckman Coulter MoFlo Astrios EQ or Cytek Aurora CS cell sorter with a 70 μm nozzle to remove poor quality nuclei and count the number of nuclei per sample. Sorted nuclei were immediately loaded on the 10x Chromium controller (10x Genomics) according to the manufacturer's protocol. Single Cell 3' v3.1 chemistry was used to process all samples and cDNA and gene expression libraries were generated according to the manufacturer's instructions (10x Genomics). HTO libraries were constructed according to the manufacturer's instructions (BioLegend). Gene expression libraries were multiplexed and sequenced on either the Nextseq 500 (Illumina) using a 75 cycle kit or the NovaSeq 6000 (Illumina) using a 100 cycle kit with the following read struture: Read 1 = 28 cycles, Index (1) = 8cycles, followed by Read 2 = 55 cycles.