GSM4561046: KLBtdTomHyp_Rep1_Panel2; Mus musculus; RNA-Seq
The method used here for generating single cell suspensions for FACs sorting was modified from a previously published protocol (Chung et. al, 2017, Nature). Brains were dissected from mice immediately following decapitation and placed in ice cold artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 20 mM NAHCO3, 20 mM dextrose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM MgCl2 which was bubbled with carbogen gas (95% O2 and 5% CO2) for at least 15 minutes prior to dissection. The hypothalamus was then dissected from each brain, and three hypothalami were pooled for each capture. The tissue was then minced and placed in 2mg/ml Pronase in ACSF and incubated at 37°C for 30 minutes. Following incubation, the supernatant was removed and tissue was resuspended in HBSS w/ 10% FBS. The tissue was then titrurated using fire polished Pasteur pipets and strained through a 70 µM cell strainer. Hoechst cell viability dye (Hoechst 33258) was added to the cell suspension at a final dilution of 4ug/ml just prior to sorting to allow for selection of living cells during FACs sorting. Cells were then FACs sorted for tdTomato fluorescence and viability via Hoechst staining. Sorting was performed on a BD FACSAriaII using a 130 µM nozzle, a sheath pressure of 10 p.s.i., and in the single cell sorting mode at the University of Iowa Flow Cytometry Facility. FACS isolated cells were then loaded on the BD Rhapsody where capture of the single cells was performed per manufacturer instructions. Following lysis of the cells and capture of mRNA on oligonucleotide barcoded beads the cDNA synthesis was performed per manufacturer instructions. cDNA libraries were then generated using two custom primer panels (with one primer panel containing lowly expressed genes and the other containing highly expressed genes with overlapping genes incorporated into each panel (Table S1 and S2) designed by BD and generated by Integrated DNA Technologies (IDT) targeted against cell type specific markers for cell types previously identified in the hypothalamus based on previous publications (Romanov et. al, Chen et. al).
GEO Accession
GSM4561046