CFTR Gene Sequencing and Genotyping

Genomic DNA was extracted from the CF-CRC cells using the QIAamp DNA Blood midi kit (Qiagen, Hilden, Germany 51183), and fluorimetric quantification was performed (Qubit, Invitrogen, CA, USA). Proximal 5’-flanking, all exons and adjacent intronic zones, and the 3′-UTR of the CFTR gene (RefSeq NM_000492.4, NG_016465.4) were sequenced using the Sanger cycle sequencing protocol (ThermoFisher Scientific, Waltham, MA, USA), as previously described [31 –33 (link)] and a genetic analyzer (ABI PRISM 3130xl; Applied Biosystems, Foster City, CA, USA). Genotype analysis was completed using multiplex ligation-dependent probe amplification (SALSA MLPA probemix P091 CFTR, MRC Holland, Amsterdam, the Netherlands, EKI-FAM, P091-100).