Illumina NovaSeq 6000 paired end sequencing; Transcriptional analysis of NPHP1 URECs
Cells were seeded (105 cells/well) in 8 wells of 96-well glass microplates and incubated for 4 days. Total mRNA was isolated using Extraction Mini Kit (Qiagen) following the recommendations of the manufacturer. cDNA synthesis from total RNA was performed using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs, Evry, France), according to the manufacturer's guidelines. Primer annealing and first strand synthesis was carried out using a reverse transcription (RT) primer mix and reverse transcriptase. “SMART” strategy by template switching was included for converting mRNA molecules into full length cDNA. Second strand synthesis and cDNA amplification were performed by DNA polymerase. The cDNA was purified using the AMPureXP Beads (Beckman Coulter, Villepinte, France) and was then sheared by enzymatic digestion. Libraries were prepared according to the manufacturer's instructions. Library quality was assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, Paris, France) and concentration measured with a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Villebon sur Yvette, France). Samples with amplification bias, identified by an unexpected library distribution on Bioanalyzer, were excluded from subsequent analysis. When samples presented a primer dimer peak in the Bioanalyzer trace, the cleanup step of the PCR reaction was repeated.