Fiber-seq of homo sapiens: adult male sorted neurons
Next, up to 5 mg input material was processed using SMRTbell kit v3.0 including repairing and A-tailing for 60 min at 37oC, 65oC for 5 min, followed by adaptor ligation at 20oC for 30 min and nuclease treatment for 15 min at 37oC, and size selected again on 2.9x AMPure PB beads. Library quality was assessed for concentration (Qubit fluorometer; high sensitivity double stranded DNA assay kit), purity (Nanodrop), size (TapeStation, Genomic DNA ScreenTape 5067-5365 and Femto pulse). Methylation status was validated with m6A slot blot assay before sequencing on the Sequel-II(e) platform, followed by deeper sequencing on Sequel II and Revio PacBio platforms for samples that passed quality controls and had sufficient material left for the additional sequencing.