GSM5286725: E6B26_B26_CTL; Mus musculus; RNA-Seq
Cell prep protocol: Dissection. P10-P12 pups were killed via decapitation and brains were removed immediately on ice. The brain was trimmed to a smaller block containing hypothalamus and was placed into a slush of EBSS. The block was then sliced on a vibratome to obtain at 1.5mm slice posterior of the anterior commissure. The slice was then trimmed under a dissection microscope to remove thalamus and cortex. Dissociation. The trimmed slices were placed immediately into papain and gently triturated ~15 times with a pipette to break up tissue before being placed on a heated (37C) orbital shaker for 30 minutes. Digested tissue was then homogenized by triturating with pipette ~ 50 times until there were no visible tissue chunks. Homogenate was spun at 500 x g for 15 minutes at room temperature. Supernatant was removed and cells were resuspended in 500uL 2% FBS in PBS. FACS. Samples were run on the aria fusion. Immediately prior to sort, cell suspension was run through a 70uM filter and washed with 500uL 2% FBS in PBS.  Cells were sorted into 8 well strip tubes and immediately spun down at 4C and frozen on dry ice. RNA from single cells was converted to cDNA using the Smart-Seq 2 protocol (Picelli, et al., Nat. Protoc., 2014). Libraries were constructed with the Nextera DNA Library Prep Kit (FC-121-1030, Illumina). Libraries were sequenced on the Illumina NextSeq500 platform using paired-end 43 bp mode.