Human induced pluripotent stem cells-derived cerebellar organoid for modelling Friedreichs ataxia disease
The short fragments (<10kb) of the HMW gDNA from the iPSC samples were removed using Short Read Eliminator (SRE) kit (Pacific Biosciences, CA). 2 ug of DNA were sheared to 14-19 kb using the Megaruptor 3 (Diagenode, Inc.) at shear speed 31. The sheared DNA were purified using 1X SMRTbell cleanup beads and used to prepare whole genome libraries using SMRTbell Prep Kit 3.0 (Pacific Biosciences, CA). Sequencing primer was annealed and Revio polymerase was bound to the library prior to loading using the Revio Polymerase kit (Pacific Biosciences, CA). Each library was loaded on one Revio SMRTcell at 200 pM loading concentration. Sequencing was performed with a 24h movie time on Revio.