NextSeq 550 paired end sequencing; Single-cell RNA-sequencing of mouse periaqueductal gray (PAG) neurons using Smart-seq2.
Single-cell RNA-sequencing of mouse periaqueductal gray (PAG) neurons using Smart-seq2.
Mouse strains are C57BL/6J mice which were injected with viral particles containing pAAV-hsyn-flex-Voltron-ST to detect the PAG cells at 10-11 weeks. Mice were sacrificed 4-5 weeks after Voltron-ST expression for voltage-imaging and single cell RNA-seq using Smart-seq2. Somas were collected by aspiration with a harvesting pipette (1.8-2.5 MΩ) containing (in mM): 90 KCl, 20 MgCl2 The harvested neuron (~0.5 μl) was ejected into a 4 μl drop of Smart-seq2 lysis buffer The RNA library was constructed according to the Smart-seq2 protocol with the following changes: SS2 cDNA was amplified for 22 cycles, 1ng of cDNA was tagmented and amplified with custom 10bp indexes