PacBio SMRT sequencing of C. elegans CB4856 (HDF5)
Mixed stage worms were collected and washed five times in M9 buffer. Worms were lysed in lysis buffer for 8 h (100 g ml-1 proteinase K, 50 mM KCl, 10 mM Tris (pH 8.3), 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween-20 and 1% beta-mercaptoethanol). DNA was extracted using phenol-chloroform extraction and ethanol precipitation. To minimize DNA shearing, we used phase-lock gel and minimized pipetting. DNA in TE buffer was treated with RNase (10 g ml-1) for 2 h and re-extracted, before being dissolved in TE buffer. Macrogen (South Korea) performed library preparation and sequencing using the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing technology (platform: PacBio RS II; chemistry: P6-C4).