Draiii

These repeat containing plasmids were cut (linearized) with restriction enzymes NheI, EcoRI-HF, BamHI-HF, or DraIII (NEB, MA, USA) (Additional file 1: Table S7) and then treated with Klenow Fragment DNA Polymerase (Takara, Shiga, Japan) at 37 °C for 30 min. The whole DNA fragments were purified using AmPureXT beads (Agilent Technologies, CA, USA), then subjected to nanopore sequencing. Library preparation was performed using a 1D native barcoding genomic DNA kit (EXP-NBD103 and SQK-LSK108) and then subjected to MinION (Oxford Nanopore Technologies) sequencing using one FLA-MIN106 (R9.4.1) flow cell according to the manufacturer’s protocol. Basecalling and fastq conversion were performed with MinKNOW ver1.11.5. De-barcoding was done using EPIME software (Oxford Nanopore Technologies).
Obtained fastq files were transformed to fasta files using seqkit fq2fa option (http://bioinf.shenwei.me/seqkit). fasta files were aligned to plasmid references like this:

lastdb -P8 -uNEAR -R01 plasmid-ref plasmid.fasta

last-train -P8 plasmid-ref reads.fasta > train.out

lastal -P8 -p train.out plasmid-ref reads.fasta | last-split > alns.maf

The unzipping template was made based on a previous method^{40 (link)}. Arm 1 DNA and arm 2 DNA have identical sequences (6.8 kbp), which were cut from plasmid pRL574. The 5′ ends of arm 1 and arm 2 were labeled with digoxigenin and biotin through PCR reaction, respectively. Each arm was cut with DraIII (NEB), leaving a 3′ overhang (CAT), and was then ligated to an adapter DNA (upper1: 5′-/5Phos/GCA GTA CCG AGC TCA TCC AAT TCT ACA TGC CGC, lower1: 5′-/5Phos/GCC TTG CAC GTG ATT ACG AGA TAT CGA TGA TTG CGG CGG CAT GTA GAA TTG GAT GAG CTC GGT ACT GCT AC for arm 1; upper2: 5′-CGT TAC GTC ATT CTA TAC ACT GTA CAG TAC, lower2: 5′-/5Phos/CTG TAC AGT GTA TAG AAT GAC GTA ACG CGC AAT CAT CGA TAT CTC GTA ATC ACG TGC AAG GCC TA for arm 2). Each ligated arm is gel purified to remove un-ligated adapters. Then, arm 1 and arm 2 were annealed via the two adapter DNAs, forming the start of the unzipping DNA segment. The rest of the unzipping segment (1.5 kbp) was cut from plasmid pRL574, at one end with AlwNI (NEB) for ligation to the start of the unzipping segment and at the other end with BstEII (NEB) for ligation to a 6T hairpin. The total length of the unzipping segment is 1515 bp without counting the hairpin (Supplementary Fig. 10). The final ligation product was gel purified using Zymo Large Fragment DNA Recovery Kit (D4045).

The mammalian expression construct containing wild-type human CACNA1A cDNA (NM_001127222.1) cloned into pMT2 LF had been published previously [27 (link)]. The deletion of exon 14 was generated by overlap extension PCR using the wild-type construct as a template and primers flanking the deletion and single restriction endonuclease sites in the CACNA1A coding region (one 5′ and one 3′ of the deletion). The primer sequences and PCR conditions are available upon request. The resulting PCR product was digested with DraIII and BmgBI (both from New England Biolabs, Ipswich, MA, USA). After gel purification, the fragment was inserted into the corresponding restriction sites of the wild-type CACNA1A construct. The resulting mutated construct was verified by Sanger sequencing.