Single cell RNA-sequencing in the suprafornical area of lateral hypothalamus

The single-cell RNA sequencing was focused on the suprafornical LHA and its surrounding areas. For single cell dissociation and collection, we used transgenic animal Agrp-IRES-Cre × Ai9 crosses, where the tdTomato-labeled AGRP neurons extend a prominent set of axonal projections to the suprafornical region of the LHA and provide a useful signal for visually guided dissection of the targeting brain region. Single-cell cDNA libraries were prepared using the Smart-SCRB chemistry. Cells from 7 male Agrp-IRES-Cre × Ai9 mice (age 6-8 weeks) were hand-sorted and cDNA libraries were prepared using the Smart-SCRB chemistry. Barcoded cDNA libraries were then pooled and sequenced on a NextSeq 550 high-output flowcell with 27 bp in read 1 to obtain the barcode and UMI, and 125 bp in read 2 for cDNA.  PhiX control library (Illumina) was spiked in at a final concentration of 15% to improve color balance in read 1. Libraries were sequenced to an average depth of 135,025 ± 38401 (mean ± S.D.) reads per cell.