Illumina HiSeq 3000 paired end sequencing; A nuclear-localized red fluorescence knock-in reporter allele for mouse pancreatic beta-cells
A nuclear-localized red fluorescence knock-in reporter allele for mouse pancreatic beta-cells
Ins2Apple/+ and MIP-GFP islets were collected from mice at age postnatal day 60. Mice were segregated by sex, and the islets from 2-3 mice of the same sex and genotype were pooled for each sample. Islets were dispersed using Accumax and FACS purified using either Apple or GFP fluorescence and either DAPI or 7AAD viability dye, respectively, to collect only live beta cells. Ins2Apple.LCA/+ mice were generated using homologous recombination into the mouse Ins2 gene locus. The mice used in this experiment (Ins2Apple/+) were generated using RMCE into the Ins2Apple/LCA allele to insert a human growth hormone minigene to improve expression of the fluorescent reporter. Ins2Apple/+ mice were backcrossed into a C57BL/6J background for at least 5 generations prior to use in experiments. MIP-GFP mice (Tg(Ins1-EGFP/GH1)1Hara) were acquired from Jackson Laboratories (stock number 006864) and were maintained in a C57BL/6J background. All mice used for experiments were fed a standard chow diet and were maintained on a 12-hour light/dark cycle. Total RNA was extracted from FACS-purified beta cells using the Maxwell simplyRNA kit (Promega). RNA was amplified using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech), and cDNA libraries were constructed using the Low Input Library Prep Kit (Clontech).
Experimental Factor: sex
female
Experimental Factor: genotype
MIP-GFP