Dnase 1 treatment

End-point RT-PCR was used to detect message for K_ATP, SK and BK channels as well as canonical endothelial or smooth muscle cell markers in FACS-purified lymphatic endothelial or smooth muscle cells from mouse. Total RNA was extracted from sorted cells using the Arcturus PicoPure RNA isolation kit (Thermo Fisher Scientific, Waltham, MA) with on-column DNase I treatment (Qiagen, Valencia, CA) according to the manufacturer's instructions. cDNA then was synthesized using the High Capacity cDNA Reverse Transcription Kit Applied Biosystems^™ (Thermo Fisher Scientific, Waltham, MA). PCR was performed in a reaction mixture containing first-strand cDNA as the template, 2 mM MgCl₂, 0.25 μM primers, 0.2 mM deoxynucleotide triphosphates; and GoTaq^® Flexi DNA polymerase (Promega, Madison, WI). The PCR program comprised an initial denaturation step at 95°C for four minutes; followed by 35 repetitions of the following cycle: denaturation (94°C, 30s), annealing (57°C, 30s) and extension (72°C, 30s). This was followed by a final elongation step for 5 min at 72°C. The Amplified PCR products were loaded on a 1.5 % agarose gel by electrophoresis, stained with SYBR-Safe (Thermo Fisher Scientific, Waltham, MA), and visualized by UV transillumination. All primers were designed to amplify intron-spanning DNA regions; primer sequences are listed in supplementary Table. 1.

For RNA isolation, frozen samples were thawed and RNA isolated according to manufacturer’s protocol before resuspension in RNase-free water. The RNA was quantified with a Nanodrop ND-1000 spectrophotometer, pooled and subjected to on-column DNase I treatment (Qiagen) and further concentration using a RNeasy-micro kit (Qiagen). RNA quality was finally assessed using a 2100 Bioanalyzer and RNA 6000 Nano kit (both Agilent).
For microarray hybridisation, 1.5μg RNA per sample (from ~300-400 isolated TEBs or ducts) was used in pooled duplicates and analysed at the Henry Wellcome Functional Genomics Facility (Glasgow). rRNA was removed using a RiboMinus Human/MouseTranscriptome Isolation kit and RiboMinus magnetic beads, labelled according to manufacturer’s protocol and finally hybridised to mouse whole-genome exon arrays (GeneChip-Mouse-Exon-1.0-ST-Array, Affymetrix UK Ltd., High Wycombe, UK) using a GeneChip Fluidics Station 450/250. The signals were measured using a GeneChip Scanner 3000 7G. CEL-files were analysed and normalised by RMA using the open-source ‘Altanalyze’ software (Emig et al., 2010 (link)). Results of differentially abundant RNAs in TEB and ducts were ranked according to raw p-value (one-way analysis of variance (ANOVA)) (Table S1). Raw data files have been submitted to GEO with the accession number GSE94371.

RNA isolation was performed with RNeasy purification including DNAseI treatment (Qiagen). Equal amounts of RNA were synthesized into cDNA using the VILO cDNA synthesis kit (Invitrogen). LuminoCt (Sigma) 2× SYBR mastermix was combined with 1 µM of both a forward and reverse primer sequence (primer sequences are listed in Supplemental Table 1) into 20 µl reactions and cycled for 95°–10 s to 60° for 30 s for 40 cycles followed by a melting curve. BioRad CFX96 was used and instrument provided software was used to determine relative normalized expression to GAPDH expression.