1 kb gene ruler

For gel electrophoresis we used 1%-broad-range-agarose (Carl Roth) gels and TAE buffer. We used the 1 kb gene ruler (Thermo Scientific; 5 μl) as a size standard. The gels were run for 120 min at 75 V at 4°C. Subsequently, the gels were removed from the gel box and placed for 20 minutes in 100 ml of 0.5 μM (1:100 000 dilution of the stock; 2 independent gels), 5 μM (1:10 000 dilution of the stock; 7 independent gels), or 50 μM (1:1000 dilution of the stock; 2 independent gels) EtBr in TAE buffer, respectively, for staining. Subsequently, the gel was de-stained in TAE buffer for 15 min. The gels were visualized using a Gel Doc XR+ system (Biorad). The same procedure was employed for SYBR Gold staining, except that the gels were stained for 20 minutes in 100 ml of 3 (1:4000 dilution of the stock; 2 gels in total) or 6 μM (1:2000 dilution of the stock; 2 gels in total). Since the dye concentration used in staining is reduced by the agarose gel matrix and the de-staining step, we used a staining correction factor of 0.1 as determined previously (19 (link)), which we use to correct all gel data.

Endothelial cells from chickens and ducks were resuspended in 350 µL RNeasy lysis buffer (Qiagen, Hilden, Germany). RNA was extracted using the RNeasy Plus kit (Qiagen, Hilden, Germany) according to the manufacturer’s guidelines. Complementary DNA (cDNA) was synthesised with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, CA, USA) and random primers according to the manufacturer’s guidelines. RT-PCR was then conducted using the Phusion high-fidelity PCR kit (Biolabs, San Diego, CA, USA) and primers specific to avian von Willebrand factor (vWBF) and CD45 (Table S1). The samples were analysed on 1% agarose gels alongside with 1kb GeneRuler (Thermo Scientific, Waltham, MD, USA).