Nanodrop photospectrometer

For RNA isolation, colon pieces were frozen in liquid N₂ and homogenized with mortar and pestle prior to resuspension in 1 mL of Trizol (Sigma-Aldrich: Cat #: 93289). RNA from cells was isolated by direct addition of Trizol reagent to the pelleted cells. Further, RNA was isolated by phenol-chloroform extraction. Shortly, 200 µL of chloroform was added, samples were shaken thoroughly and centrifuged (13000 rpm for 15 min at 4 °C) and subsequently RNA from the upper aqueous phase was precipitated with 700 µL isopropanol and 200 µg of glycogen to enhance precipitation. After pelleting, RNA was washed with 75 % ethanol and air-dried at 56 °C in a thermoshaker. Depending on the size, the pellet was reconstituted in 10-50 µL of DEPC-treated water (Life Technologies) and dissolved at 56 °C for 10 min. RNA concentration was determined with a NanoDrop photospectrometer (Thermo Scientific).

Skin biopsies were collected from 25 SSc patients after informed consent study number: NL57997.091.16. RNA was extracted using RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Next, RNA concentration was measured using a Nanodrop photospectrometer (Thermo Scientific, Waltham, MA, USA), and subsequently, 1 μg of RNA was converted into cDNA in a single step reverse transcriptase PCR using oligo dT primer and M-MLV Reverse transcriptase (Life Technologies, Carlsbad, CA, USA). In this cDNA, FAP gene expression was measured using 0.2 µM of validated cDNA-specific primers (see Table 1) (Biolegio, Nijmegen, the Netherlands) in a quantitative real time polymerase chain reaction (qPCR) using SYBR green master mix (Applied Biosystems, Waltham, MA, USA). Relative gene expression (−ΔCt) was calculated using four reference genes: GAPDH, HPRT, TBP, and RPS27A.

Venom samples were redissolved in LCMS water and diluted to a concentration of 0.18 mg/mL. We determined venom protein concentration by the A280 reading from a NanoDrop Photospectrometer (ThermoFisher Scientific, Waltham, MA, USA). We then loaded 15 μg of venom protein onto a Prominence HPLC apparatus (Shimadzu Corp., Columbia, MD, USA). Samples were run through a Phenomenex Aeris widepore column (3.6 micron, C18, part # 00G-4482-AN) maintained at 25 °C. Samples were eluted with solution A as 0.1 mM TFA in water and solution B as 0.06 mM TFA in acetonitrile, and absorbance at 220 nm was measured over 150 min with the following run parameters: 5 min at 10% B, 110 min increasing from 10 to 55% B, 5 min increasing from 55 to 75% B, 5 min at 75% B, 5 min decreasing from 75–10% B, and 5 min at 10% B. Venom peak areas were quantified using Shimadzu Lab Solution v2 software, combining default peak calls by the iPeakFinder algorithm with manual curation of the peak baseline and filtering of peaks making up less than 0.1% total area as noise. Peaks were called uniquely per individual snake, by aligning the serial HPLC profiles from all samples from a single individual and calling peaks that were present. The relative abundance of HPLC peaks in each sample will be henceforth referred to as venom composition.