title: GSM8098631: dcm-/dam- lambda phage DNA, M.SssI [LMD_mdC]; Escherichia phage Lambda; OTHER
library_construction_protocol: Lambda DNA dcm-/dam- was purchased from Thermo Fisher, catalog number: SD0021 Libraries for the Sequel IIe were prepared according to PacBio's SMRTbell express template prep kit 2.0 (PN: 100-938-900). Briefly, for DNA-repair and A-tailing, 300 ng - 1 µg DNA (1 kb) in 46 µl nuclease free water, 8 µl repair buffer, 4 µl end repair mix and 2 µl DNA repair mix were mixed by pipetting and incubated at 30°C for 30 min. The reaction was inactivated by incubating at 65°C for 5min. Next, 4 µl SMRTbell barcoded adapter (Barcoded overhang adapter kit 8B, PN: 101-628-500), 30 µl ligation mix and 1 µl ligation enhancer were mixed by pipetting. 31µl of the ligation mix was directly added to the A-tailed DNA. The reaction was incubated for 30 min at 20°C and subsequently purified using SMRTbell cleanup beads. Non-SMRTbell-DNA was depleted by nuclease treatment. The concentration of the final library was determined using the Qubit dsDNA-HS-Assay-Kit (ThermoFisher: cat. no. Q32851).