mRNA was isolated using RNeasy Mini kit from Qiagen following manufacturer’s instructions. RNA was treated with DNase I (1 unit for 1µg RNA; Roche) following which the DNase was inactivated by EDTA (final concentration of 3mM) at 65°C for 10 min. High capacity RNA-cDNA kit (Applied biosystems catlog # 4387406) was used to make cDNA from RNA, following manufacturers instructions. Gene expression levels were measured by quantitative real-time PCR (qPCR) using Taqman gene expression assays (The Step One Plus Real-Time PCR System; Applied Biosystems, Carlsbad, CA). Gene expression assays used were: Mm00626012_m1 (SLC9A9 solute carrier family 9 (sodium/hydrogen exchanger), member 9), Mm00555445_m1 (SLC9A6 solute carrier family 9 (sodium/hydrogen exchanger), member 6). Mm03928990_g1 (Rn18s, 18S ribosomal RNA) and Mm99999915_g1 (GAPDH, glyceraldehyde-3-phosphate dehydrogenase) were our endogenous control. Each experiment had three technical replicates and was repeated three times independently (biological replicates) to account for intra- and inter-assay variances respectively. Ct values were used for all manipulations, and were first normalized to endogenous control levels by calculating the ΔCt for each sample. Values were then calculated relative to control to generate a ΔΔCt
