Ampure xp bead purification

NimaGen CFTR‐HS kit (version 0.3) was designed in collaboration with the manufacturer (NimaGen, The Netherlands). The analysis was performed following manufacturer's protocol: 20–80 ng maternal gDNA or pooled fetal WGA‐DNA was input for Reverse Complement‐PCR, where sample‐specific indexes and P5/P7 sequences (NimaGen) were added to create an NGS‐library for Illumina‐based sequencing with a mean insert size of 250 bp. Following equal volume pooling of the libraries and AMPure XP bead purification (Beckman Coulter), the libraries were sequenced on a MiniSeq (Illumina) in a 1x100 bp rapid run. Bcl2fastq and demultiplexing were performed using Local Run Manager (Illumina). The FASTQ files were aligned to the hg38 reference genome without alternate contigs²⁷ using bwa mem (version 0.7.17) and the resulting BAM and VCF files were explored in Integrative Genomics Viewer (Broad Institute, UC San Diego).
The bioinformatic pipeline used SnpAhoy 0.5.2²⁸ for calling the genotypes on 48 CFTR‐related SNP‐positions. SNP‐positions with genotype calls differing from the hg38 reference genome were reported as possible variants. The common 3‐base deletion F508del was detected using Freebayes 1.3.6²⁹, ³⁰ while the 21kb deletion CFTRdele2,3 was detected using Delly 0.9.1.³¹, ³² Filtering of the vcf‐file from Freebayes was done using VCFtools 0.1.16.³³, ³⁴

Complementary DNAs (cDNAs) were prepared according to Briese et al. (48 (link)). Briefly, RNA was extracted from hamster swabs and tissues following the QiaAmp Viral RNA extraction protocol (Qiagen), and 11 μl was taken into the SuperScript IV First-Strand cDNA synthesis system (Thermo Fisher Scientific) following the manufacturer’s instructions. After ribonuclease H treatment, second-strand synthesis was performed using Klenow fragment (New England Biolabs) following the manufacturer’s instructions. The resulting double-stranded cDNAs (ds-cDNA) were then purified using Ampure XP bead purification (Beckman Coulter) and eluted into 30 μl of water.

2ug total RNA was used for cDNA library preparation by using a modified protocol based on the Illumina Truseq RNA Sample Preparation Kit V2. After poly-A selection, fragmentation, and priming, reverse transcription was carried out for 1^st strand cDNA synthesis in the presence of RNaseOut (Invitrogen) and actinomycin-D (MP Biomedicals). The synthesized cDNA was further purified by using AMPure RNAClean beads (Beckman Coulter). A modified method by incorporation of dUTP instead of dTTP was prepared and used for the second strand synthesis. After AMPure XP bead purification (Beckman Coulter), following the standard protocol recommended by the Illumina Truseq RNA kit, end repairing, A-tailing, and ligation of index adaptors were sequentially performed for generation of cDNA libraries. After size selection of libraries using Pippin Prep (SAGE Biosciences), the dUTP-containing strands were destroyed by digestion with USER enzymes (New England Biolabs) followed by PCR enrichment. Final cDNA libraries were analyzed in Agilent Bioanalyzer and quantified by Quant-iT Pico-Green assays (Life Technologies) before sequencing using the HiSeq platform (Illumina).