GSM4223659: WT pooled; Mus musculus; RNA-Seq
Micro-dissected arcuate nucleus and median eminence were dissociated to single-cell suspensions. Live cells were sorted and processed through Drop-seq with flow rates of 3000 μL/hr (cells and beads) and 13,000 μL/hr (oil) were used, and beads were resuspended at 167,000 per mL. Droplets were broken with perfluorooctanol in SSC to release beads. The beads were washed and resuspended in a reverse transcriptase mix, followed by a treatment with exonuclease I to remove unextended primers. The beads were then counted and PCR amplified. The amplified cDNA was fragmented and amplified for sequencing with the Nextera XT DNA sample prep kit (Illumina) using custom primers that enabled the specific amplification of only the 3' ends (described in Macosko et al. Cell 2015).