SCA3 sam012
Genome DNA was obtained from peripheral blood from 20 unrelated SCA3/MJD affected and 5 unaffected individuals by standard procedures. Target sequences including the CAG repeats fragment (about 1.6k) were amplified with two specified primers (c: GATTCTCGGATTTAGGATGC; f: ATAAAGTGTGAAGGTAGCGAAC). Amplification conditions were as following: about 50 ng DNA template for each sample was added to a 25 ul mastermix containing 5 ul 5X PrimeSTAR GXL Buffer, 5 mM dNTP Mixture, 7.5 mM primers, 0.65 U PrimeSTAR GXL DNA polymerase and 15 ul Water ddH2O. Samples were amplified with an initial heat denature step of 95 C  for 5 minutes, followed by 35 cycles of 98 C for 10 seconds, 68 C for 2 min sec and Final Extension of 68 C for 10 minutes, and 4 C for hold. After barcoded and purified by magnetic bead, PCR products with equal molar ratio were pooled and constructed as a SMRTbell library following a standard protocol (SMRTbell Template Prep Kit 1.0). The annealed SMRTbell templates were bounded with DNA polymerase enzymes using the DNA Polymerase Binding Kit, and incubated with 9 nM of polymerase in the presence of phospholinked (Pacific Biosciences) nucleotides for 6 hours at 30 C. After that, the library is stored at 4 C. Sequencing was performed within 36 hours of binding. The library was sequenced as described in Pacbio official document using commercial sequencing chemistry. Sequencing data collection was performed on the PacBio Sequel (Pacific Biosciences) for 2 hours.