Grouped ensemble coding of behavioral states by combinations of molecularly defined cell types

We used a plate-based method (SMART-SCRB) to transcriptionally profile manually picked single neurons from the paraventricular nucleus of hypothalamus (PVH) from 10 male Agrp-IRES-Cre crossed with Ai9 reporter mice, all >7 weeks old. For each mouse, the PVH was dissected and cells expressing the tdTomato reporter were manually selected and placed into wells of a series of 96-well plates. We used a plate-based method with cell and transcript barcording and reverse transcription in each well, followed by pooling and amplification using a modified SMART-Seq technology that yields digital 3' end counts of transcripts. We processed a total of 8 96-well plates, of which some wells were deliberately left empty. External RNA controls (ERCCs) were spiked into each well to allow for QC and accurate transcript quantification.