GSM5076376: Sim1td-Cont pooled; Mus musculus; RNA-Seq
For Sim1-Cre;Ai14 tdTomato cell sorting, pups (P1-P4) were dissected, then hypothalami were rapidly extracted. Pooled hypothalamus samples from 5-6 mice were digested by papain solution (Worthington Biochemical) with DNase in Hibernate A media for 30 minutes at 37 °C with gentle agitation. Digestion was stopped by dilution with Hibernate A containing B27 (Thermo), Glutamax (Thermo) and 0.5% Trehalose (Sigma-Aldrich), then gently triturated using a series of three Pasteur pipettes with tips fire-polished to incrementally smaller openings. Cell suspension was centrifuged at 300g for 5 min, resuspended in Hank's balanced salt solution (HBSS) with 0.5% BSA and 0.5% Trehalose. Sytox blue (Thermo, 1:1000) was added to the cell suspension, and cells were filtered a second time through a 70-m cell strainer. Viable (Sytox blue-negative) tdTomato+ cells were subsequently sorted using a MoFlo Astrios (Beckman Coulter) cell sorter. Following cell sorting, cells were pelleted at 400g for 5 min, re-suspended in PBS with 0.01% BSA, and counted with a hemocytometer. Cells were adjusted to a final concentration of 140,000 cells/mL. Drop-seq was then carried out at the Princess Margaret Genomics Facility. 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).