Sp6 or t7 polymerase

Whole mount in situ hybridization was performed according to standard protocols. The SP6 or T7 polymerase (NEB, Germany) along with the DIG RNA labelling system (Roche, Germany) were used to in vitro transcribe probes from linearised plasmids. To detect charon transcripts a 583 bp fragment of the complementary DNA (cDNA; NM_212969) was cloned by TOPO TA cloning into pCRII, from which a DIG-labelled probe was transcribed after linearization. All other probes used in this project have been described before: cardiac myosin light chain 2 (cmlc2), chondroitin sulphate proteoglycane 2a (cspg2a), insulin (ins), lefty2, notch1b, nkx2.5, pitx2^{65 (link)} and tbx2b^{66 (link),67 (link)}.

A 20μg aliquot of total cellular RNA was separated on a 1% Seakem GTG agarose gel as previously described [90 (link)]. Three to six μg of poly A⁺ RNA was separated on an 0.8% Seakem GTG agarose gel as previously described [90 (link)]. Separated RNA was transferred to a Hybond XL membrane (GE Healthcare) using a gradient of 6X SSC to 10X SSC overnight at room temperature. Synthesis of ³²P-labeled RNA riboprobes was carried out in vitro using SP6 or T7 polymerase (New England Biolabs). Membranes were incubated with probes in NorthernMax PreHyb Buffer (Ambion) at 65°C overnight. Images were scanned using a Typhoon 9400 scanner, and quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).