GSM2304152: S21_BH10; Mus musculus; RNA-Seq
Single sorted beta cells were captured on a medium-sized (10–17 mm cell diameter) microfluidic RNA-seq chip (Fluidigm) using the FluidigmC1 system. Cells were loaded onto the chip at a concentration of 250 cells/ml, stained for viability (LIVE/DEAD cell viability assay; Molecular Probes, Life Technologies) and imaged by Zeiss Axio Oberver Z1 microscope to assess the number and viability of cells per capture site. Only single, live cells were included in the analysis. Cell lysis, cDNA synthesis, and PCR reactions were performed on the same chip using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Clonetech). For each C1 experiment, two bulk RNA controls (approximately 250 cells/sample) were processed in parallel in PCR tubes, using the same reagent mixes as used on chip as described in Fluidigm protocol 100-5950. Libraries were prepared using the Illumina Nextera XT DNA sample preparation kit