Illumina HiSeq 4000 sequencing; Single cell RNA-seq of individual cells from dorsal skin of a F1 mouse cross-breed (C57BL/6JxCAST/EiJ)
Single cell RNA-seq of individual cells from dorsal skin of a F1 mouse cross-breed (C57BL/6JxCAST/EiJ)
Skin tissue was dissected from 9 week old female F1 offspring of matings between CAST/EiJ and C57BL/6J mice. Cells were dissociated from skin as described in Joost et al., or using GentleMACS (Miltenyi Biotec); with both methods giving similar cell yields and viability. Briefly, for the GentleMACS method, dorsal skin was cut and minced into small pieces (approximately 1x1mm) and incubated in HBSS (Sigma) + 0.04% BSA (Sigma) + 0.2% Collagenase Ia (Sigma) at 37°C for 60 minutes with occasional agitation. Thereafter this slurry was processed on a GentleMACS (Miltenyi Biotec) with 2x Program D, cell-strained (70um) and washed. Residual tissue was further treated with HBSS + 0.05% Trypsin-EDTA (Sigma) at 37°C for 15 minutes and processed likewise. For the Joost et al method, GentleMACS dissociation was substituted with manual disaggregation by smashing tissue fragments against a cell strainer with the piston from a 5 mL syringe Cell lysis during sorting by FACS. Cells were subjected to the Smart-seq2 protocol with no modification.