Purelink hipure plasmid maxiprep kit

BAC clones were identified by PCR from a library prepared by the Clemson University Genomics Institute. Briefly, clones from 12 microtiter plates (386 well) were pooled to form superpools, DNA was isolated for each superpool using the PureLink HI Pure Plasmid Maxiprep Kit (Invitrogen), and each superpool was screened by PCR using primers designed to amplify fragments from edn3 and tyr DNA sequences (S7), using PCR conditions described above for amplifying bacterial colonies. The location of positive clones within microtiter plates of a superpool was determined by sequential PCR of plate, column, and row BAC pools. BAC DNA for positive PCR reactions, as visualized on agarose gels, was isolated using the PureLink HI Pure Plasmid Maxiprep Kit (Invitrogen) and then sequenced to 30x depth on a PacBio RS II by the Duke Center for Genomic and Computational Biology. Resulting reads were assembled using the SMRT Analyses 2.1 HGAP pipeline^{58 (link)}.

Two Mission short hairpin RNA (shRNA), one targeting the 3′UTR of the gene coding for ENO1 (TRCN0000029324) and one targeting the CDS region (TRCN0000029327) were used to transform bacteria (Sigma-Aldrich, Milan, Italy); plasmids were purified with the PureLink HiPure Plasmid Maxiprep Kit (LifeTechnologies). Lentiviruses were produced by co-transfecting 293T packaging cells (Clontech by Diatech Lab Line Srl, Jesi, AN, Italy) with pLKO.1 puro vector containing the shRNA and the helper vectors pCMVΔ8.74 (Add gene, Cambridge, MA, USA) and pVSV-G (Clontech), using the calcium phosphate method. Lentiviruses collected at 24h after transfection were used for the transduction of the CFPAC-1 cell line supplemented with 8 μg/ml polybrene (Sigma-Aldrich) and, after 48h of infection, cells were then selected for stable silencing using 2 μg/ml Puromycin (Sigma-Aldrich). For quantitative mRNA expression analysis, a polymerase chain reaction (PCR) was carried out with total cDNA and the SYBR Green PCR Master Mix (LifeTechnologies), with a two-step amplification protocol. mRNA expression of target genes was normalized using the mRNA level of β-Actin.