Pacbio SEQ2 Short insert library: HEK 293 with transfection of an empty pCI plasmid (HEK-pCI)
HEK 293 cells were maintained by the procedures of Bulanenkova et al., 2007. T25 flasks with approximately 10^6 HEK 293 cells were transfected using Lipofectamine 3000 (Invitrogen, cat#L3000015, Carlsbad, CA, USA) according to manufacturer's recommendations. Briefly, immediately before transfection, media was changed on the flask. Lipofectamine:DNA complexes for the pCI null vector (Promega, cat#E1731, Madison, WI, USA) were added to the media with 6.5ug DNA administered. Lipofectamine:DNA complexes were prepared in OptiMEM medium (Gibco, cat#31985062, Gaithersburg, MD, USA), then added to HEK growth media (RPMI 1640:DMEM:F-12 (2:1:1) medium supplemented with 10% fetal bovine serum [Gibco: RPMI-1640: cat#11875093; DMEM/F12: cat#11320033; FBS: cat#A3160402]) currently in the flask. Following transfection, flasks were returned to incubate at 37 C /5% CO2. At 48 hrs post infection, the transfection medium was replaced with fresh HEK growth medium in each flask. At 72 hrs post infection, flasks were harvested by scraping cells in PBS, and DNA was isolated by a Wizard Genomic DNA Purification Kit (Promega, cat#A1120). The short-insert DNA SMRTbell library was performed according to the manufacturers instructions by first shearing the sample to ~200bp using a Covaris LE220 Focused-ultrasonicator,followed by damage and end repair and ligation of single-stranded barcoded adaptors. Sequencing primer was then annealed to the SMRTbells and incubated with the template for 1 hour at 20 C using a ratio of 20:1. The Sequel 2.1 polymerase was then bound to the annealed template complex using a ratio of 30:1 polymerase to SMRTbell at 0.5 nM for 4 h at 30 C. The binding complex was cleaned up, diluted to the final loading concentration, and held at 4 C until diffusion-based loading on the Sequel II instrument. Diluted binding complex at concentration of 125-175pM was loaded on the 8M sequencing chip and 30 hour movies were recorded according to the manufacturers guidelines.