Kh2po4

During the isolated heart experiments, after thoracotomies performed under ketamine/xylazine anaesthesia, the brain was removed and placed into a silicone containing petri dish, filled with 0–4 °C Krebs solution (composition in mmol: 110 NaCl, 5.0 KCl, 1.0 MgSO₄, 1.0 KH₂PO₄, 5.0 glucose and 24.0 NaHCO₃, obtained from Sigma-Aldrich, St. Louis, MO, USA). The solution was equilibrated previously with a gaseous mixture of 5% CO₂, 10% O₂ and 85% N₂ at pH 7.4. Basilar arteries were isolated with microsurgical tools (Fine Science Tools GmbH, Heidelberg, Germany). The arteries were equally cut into 4 mm long rings, which were then mounted in an isometric contraction measurement system (DMT-510, Danish Myotechnology, Aarhus, Denmark). The Ca²⁺-free Krebs solution was changed to Ca²⁺-containing one (composition in mM: 110 NaCl, 2.5 CaCl_2, 5.0 KCl, 1.0 MgSO₄, 1.0 KH₂PO₄, 5.0 glucose and 24.0 NaHCO₃, obtained from Sigma-Aldrich, St. Louis, MO, USA). Before every experiment, a normalization protocol was performed, by stretching the preparations with 1.5 mN force, which was increasing evenly every 15 s until the calculated intraluminar pressure reached 13.4 kPa. The experiments were then performed at this stretch level. Contractile responses for KCl (6–66 mM), serotonine (1 nM–10 μM) and angiotensin II (1 nM–100 μM) have been recorded.

The labeled peptides were reconstituted in 10% formic acid prior to fractionation using strong cation exchange (SCX) as described previously (Helbig et al, 2010 (link)) for the protein expression levels analysis. The SCX system consisted of an Agilent 1100 HPLC system (Agilent Technologies, Waldbronn, Germany) with two C₁₈ Opti-Lynx (Optimized Technologies, OR) trapping cartridges and a polysulfoethyl A SCX column (PolyLC, Columbia, MD; 200 mm × 2.1 mm inner diameter, 5 μm, 200-A). The peptides were dissolved in 10% FA and loaded onto the trap columns at 100 μl/min and subsequently eluted onto the SCX column with 80% acetonitrile (ACN; Biosolve, the Netherlands) and 0.05% FA. SCX buffer A was made of 5 mM KH₂PO₄ (Merck, Germany), 30% ACN and 0.05% FA, pH 2.7; SCX buffer B consisted of 350 mM KCl (Merck, Germany), 5 mM KH₂PO₄, 30% ACN and 0.05% FA, pH 2.7. The gradient was performed as follows: 0% B for 10 min, 0–85% B in 35 min, 85–100% B in 6 min and 100% B for 4 min. After injection of 200 μg of labeled lysate, a total of 45 fractions were collected, dried in a vacuum centrifuge and stored at −80°C.

Healthy adult Suffolk sheep (n = 6) were bled by puncture of the jugular vein and peripheral blood was collected in BD Vacutainer® heparin tubes (Franklin Lakes, USA). Approximately 20 mL of heparinized blood were diluted in 20 mL sterile PBS with 0.02% EDTA (Sigma-Aldrich, St. Louis, USA), layered on top of 12 mL Biocoll® separating solution (density = 1.077 g/L; Sigma-Aldrich) and centrifuged (800 × g, 45 min). After removal of plasma and peripheral blood mononuclear cells (PBMC), the cell pellet was suspended in sterile Hanks’ Balanced Salt Solution 1X (HBSS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) for subsequent lysis of ovine erythrocytes by using lysis buffer solution (0.3 mM NaH₂PO₄, 0.4 mM KH₂PO₄, all Merck, Burlington, Massachusetts, USA) for 1 min at room temperature (RT). Then, a hypertonic buffer [0.3 mM NaH₂PO₄, 0.4 mM KH2PO4, 0.136 mM NaCl (all Merck)] and HBSS were added to the solution and centrifuged at 600 × g for 10 min. This process was repeated twice. Finally, the pellet was washed in sterile HBSS solution at 600×g for 10 min two times. PMN were re-suspended in sterile HBSS medium (Gibco), counted in a Neubauer haemocytometer chamber, and left on ice to rest (30 min) before use.

Inside the anaerobic chamber (Bactron VI, Shellab, Sheldon Manufacturing, Incorporation using a mixed gas of 5% hydrogen, 5% carbon dioxide, and 90% nitrogen) at 38 °C a volume of 100 μL of mix culture of SRB was inoculated and enriched in culture standard medium. The Postgate standard medium was used for the growth of the SRB; for the maintenance a sulfate poor Postgate medium, agar was used and it is referred as the modified Postgate medium. Sodium lactate was used as a carbon source. Blackening of the medium due to the formation of iron sulfides was indicative of positive growth.
The Postgate standard medium contained per L: agar, 2.0 g (Difico); KH₂PO₄, 0.5 g (Merk); NH₄Cl, 1.0 g (J.T. Backer); Na₂SO₄, 1.0 g (Merk); CaCl₂, 1.0 g (Vetec); MgCl₂·6H₂O, 1.83 g (Merk); yeast extract, 1.0 g (Merk); ascorbic acid, 0.1 g (Merk); sodium thioglycolate, 0.013 g (Vetec); sodium citrate, 6.38 g (Synth); sodium lactate 1.75 mL; NaCl (Quemis) 3.5%, resazurin, 2.0 mL (Vetec) 0.025% P/V, FeSO₄·7H₂O, 0.5 g (Merk). All the components were dissolved in distilled water and the pH was adjusted to 7.5–8.0 using 5 M HCl. After this, the solution was homogenized by agitation and later sterilized at 121 °C for 30 min.

4C-seq was performed as previously described^{88 (link)}, with minor alterations. Whole pancreas was dissected from 6 to 12 adult zebrafish (7–15 × 10⁶ cells; both genders and with 12–24 months), kept on ice in PBS [137 mM NaCl (#S/3161/60, Fisher Chemical), 2.7 mM KCl (#2676.298, VWR), 10 mM NaHPO4 (#1.06342.0250, Merk), and 1.8 mM KH2PO4 (#1.06585.1000, Merk)] with 1x Complete Proteinase Inhibitor (#11697498001, Roche), fixed in 2% formaldehyde (#F1635-500ML, Sigma-Aldrich) for 10 min, and stored at −80 °C. Cell lysis was performed on ice, with a 15 mL Tenbroeck Homogenizer, not exceeding 10 min. Ligation was performed with 60U T4 DNA Ligase (#EL0012, ThermoFisher Scientific). The restriction enzymes used were DpnII (#R0543M, NEB) and Csp6I (#ER0211, ThermoFisher Scientific) for the first and second cuts, respectively. Chromatin was purified by Amicon Ultra-15 Centrifugal Filter Device (#UFC901024, Milipore). 4 C libraries were prepared for Illumina sequencing by the Expand Long Template Polymerase (#11759060001, Roche) with primers targeting the TSSs of each gene and including Illumina adapters (Supplementary Dataset 4c). Final PCR products were purified with the High Pure PCR Product Purification Kit (#11796828001, Roche) and AMPure XP PCR purification kit (#B37419AB, Agencourt AMPure XP).

The whole pancreases were dissected from double transgenic adult zebrafish [Tg(ins:GFP, ela:mCherry), Tg(ins:GFP, gcga:mCherry), and Tg(ins:GFP, sst:mCherry)] and fixed using 4% formaldehyde (#F1635, Sigma-Aldrich) in 1xPBS [137 mM NaCl (#S/3161/60, Fisher Chemical), 2.7 mM KCl (#2676.298, VWR), 10 mM NaHPO4 (#1.06342.0250, Merk), and 1.8 mM KH2PO4 (#1.06585.1000, Merk)]. Cells were dissociated, on ice, using a 15 mL Dounce homogenizer in 1 mL of ice-cold sort buffer [1% EDTA (#20301.290, VWR), 2 mM HEPES (#83264, Sigma-Aldrich) pH 7.0 in 1xPBS), and then passed through a 40-μm cell strainer. Immediately following dissociation, the mCherry and GFP fluorescence were analysed on a BD FACS-ARIATM II cell sorter (BD Biosciences).