Dark-tolerant Prochlorococcus NATL2A, after seventh transfer replicate 2, PacBio WGS
Culture samples were prepared by pelleting 90 mL of exponentially growing cultures by centrifugation at 7,500 xg for 15 minutes. The pellet was resuspended into 200 l of pH 8 TE and stored at -80C. DNA was extracted using the same method as previously described above. Library construction began with diluting 4g of intact genomic DNA in 150 l of TE buffer (10 mM TrisHCl, 1 mM EDTA, pH 8.5) and fragmenting to 10-12 kb lengths using a gTube (Covaris Cat# 520079). Fragmented DNA was concentrated by a 0.45X SPRI bead cleanup (Beckman-Coulter), eluted in 37 l EB (10 mM TrisHCl pH 8.0) and processed using SMRTBellTM Template Prep kit 1.0 (PacBio Part# 100-259-100) to SMRTbell libraries, following manufacturer guidelines. Indexed blunt-end SMRTbell adapters (PacBio Part# 100-4666-000) were added for multiplexing. Multiple indexed SMRTbell libraries were assembled in a single pool, cleaned up using a Minelute reaction cleanup kit (QIAGEN Cat# 28204) to 100 l volume in EB, followed by SPRI 0.4X cleanup, with final elution in 11 l EB. Concentration and quality were assessed by Picogreen (ThermoFisher QuantIt Cat# P11496) and FEMTOpulse (Agilent). Pooled libraries were bound using Sequel Binding Kit 2.1 and sequenced for 10 h on the Pacific Biosciences Sequel I using a SMRTCell 1Mv2.