Splicing Minigene Construct Generation

EGFP-MBNL1-41, EGFP-MBNL2-38, Atp2a1-WT and Atp2a1-D minigenes were previously described^{17 (link), 57 (link)}. Mouse Ank2 and mutAnk2 as well as human ANK2 and mutANK2 splicing minigenes were generated by cloning DNA oligonucleotides between NotI and SalI restriction sites in Atp2a1-D minigene. 120 bp DNA oligonucleotides contained selected TGCT(N)₃TGCT(N)_13–18TGCT/C or mutated GGCT(N)₃TGAT(N)_13–18TGTC sequences (substitutions are underlined) as well as NotI and SalI restriction sites at 5’ and 3’ ends respectively. DNA oligonucleotide sequences are listed in the key resources table. The complementary single-stranded DNA oligonucleotides (100 μM) were annealed in Annealing Buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA) at 95°C for 5 minutes followed by cooling to 25°C for 45 minutes. Annealed oligonucleotides were digested with NotI and SalI restriction enzymes (New England Biolabs), purified using Clean-Up Concentrator Kit (A&A Biotechnology), and ligated. The design of the hybrid Atp2a1 minigenes preserves RNA structures within a thermodynamically stable region at the Atp2a1 insertion site^{57 (link)}. Final splicing minigenes were tested by Sanger sequencing.