Superscript 3 supermix

To quantify NDC80^luti mRNA levels as described in Figure 4—figure supplement 1A, we used a reverse transcription combined with quantitative PCR (RT-qPCR) protocol. Total RNA was isolated, purified and treated with DNAse (Macherey-Nagel (Düren, Germany)). 750 ng of total RNA was reverse-transcribed using random primers and Protoscript II (NEB (MA, USA)), and single-stranded cDNA was quantified by real-time PCR using SYBR green mix (Life Technologies). The signals were normalized to ACT1 transcript levels. The oligonucleotide sequences used for RT-PCR experiments are displayed in Supplementary file 2.
For the RT-qPCR in Figure 5—figure supplement 1A, RNA was isolated by acid phenol-chloroform extraction, treated with DNAse (TURBO DNA-free kit, Thermo Fisher (MA, USA)), and reverse transcribed into cDNA (Superscript III Supermix, Thermo Fisher). The cDNA was quantified using the Absolute Blue qPCR Mix (Thermo Fisher). The NDC80^luti signals were normalized to ACT1 transcript levels. The oligonucleotide sequences used for RT-qPCR experiments are displayed in Supplementary file 2.

Total RNA was isolated using TRIzol (Life Technologies) according to a previously described protocol (Bogart and Andrews 2006). 450 ng of isolated RNA was treated with DNase (TURBO DNA-free kit, Thermo Fisher) and reverse transcribed into cDNA (Superscript III Supermix, Thermo Fisher) according to the manufacturer’s instructions. The RNA levels of specific Adh isoforms were quantified using primers specific to Adh^DIST and Adh^PROX (Table 1, supplemental file 1), SYBR Green/Rox (Thermo Fisher), and the StepOnePlus Real-time PCR system (Thermo Fisher). Adh^DIST and Adh^PROX signals were normalized to αTUB84B transcript levels. RT-qPCR for each sample was performed in technical triplicate and the mean Ct value was used for the normalizations. The efficiency value for each oligonucleotide pair was empirically determined and only those pairs that had greater than 90% efficiency were used for the RT-qPCR experiments. The oligonucleotide sequences used for the RT-qPCR experiments are displayed in Table 1, and primer efficiency calculations are shown in supplemental file 1. The raw Ct values and analyses for all the qPCR experiments are shown in supplemental files 2 through 6.