Direct zol kit

For murine ear tissue, epidermal ear sheets were separated and floated on trypsin GNK for 15 min at 37°C. Then, two curved forceps were used to separate the epidermis from the dermis of each ear sheet, and both layers of skin were then homogenized using scissors and placed into TRIzol for quantitative PCR (qPCR) analysis. Total RNA was isolated using a Direct-zol Kit (Genesee Scientific, Research Triangle Park, NC) or using TRIzol reagent (Invitrogen). RNA was reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA), and the resulting cDNA was amplified using Fast SYBR Green Master Mix (Applied Biosystems). Primers for amplification are listed in table S1 online. Fold induction of gene expression was normalized to the house keeping glyceraldehyde-3-phosphate dehydrogenase and calculated using the 2(−ΔΔCt) method.

Total RNA was isolated from tissue and cells using a Directzol kit (Genesee Scientific; El Cajon, CA). The gene expression was measured using TaqMan probes (Applied Bio-systems, Foster City, CA) or SYBR Green probes (Thermo Fisher Scientific) on LightCycler96 (Roche Diagnostics, Indianapolis, IN). Liver NHE8, tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, and Lgr5 were measured using the TaqMan reaction system. Collagen 1α (Col 1α) was measured using the SYBR Green system. TATA-binding protein (for TaqMan probes) and β-actin (for SYBR Green probes) were used as an endogenous reference to normalize gene expression levels.