Experion automated electrophoresis

Frozen lice were homogenized using 5 mm stainless-steel beads and a Tissue-lyser (Qiagen). RNA was extracted using TRIzol (Invitrogen) following manufacturer’s instructions with modifications. Specifically, following the organic phase extraction, the supernatant was removed and RNA was then purified using RNeasy spin columns (Qiagen) with an on-column DNase I digestion to remove genomic DNA as per manufacturers’ instruction. Total RNA was eluted in 30 μL ultra-pure water and quantified by spectrophotometry (Nanodrop-1000, Thermo Fisher). RNA quality was determined using Experion Automated Electrophoresis (Bio-Rad) with all samples having an RQI < 9.

Total RNA was extracted from INS1 832/13 and EndoC-βH1 cells using RNAeasy (Qiagen, Hilden, Germany) before complementary DNA (cDNA) was synthesized using SuperScript (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. Concentration and purity of total RNA were measured with a NanoDrop ND-1000 spectrophotometer (A260/A280 > 1.9 and A260/A23 0 > 1.4) (NanoDrop Technologies LLC, Wilmington, DE, USA). RNA Quality Indicator (RQI) higher than 8.0 (Experion Automated Electrophoresis, Bio-Rad, USA) was considered to be high-quality total RNA preparation. TaqMan mastermix from Applied Biosystems (Foster City, CA, USA) was used for qPCR and performed following manufacturer’s protocol and was run in a 7900 HT Fast Real-Time System (Applied Biosystems). The qPCR was carried out as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. Changes in gene expression were calculated using the ΔΔCt method with a fold-change cut-off at ≥ 1.5 and p < 0.05 considered significant. All samples were run in duplicate, and relevant negative controls were run on each plate. qPCR results were normalized to housekeeping genes (PPIA or HPRT). Primer sequences used in the qPCR assays are provided in Supplementary Table S2.