Bioanalyzer chips

The RNA quality was assessed by Bioanalyzer chips (Agilent) using buffers and instructions provided by the manufacturer.

The quantity of LR-PCR DNA templates (prior and during library preparation) was assessed by spectrophotometry (Nanodrop ND-800, Thermo Scientific, Waltham, MA, USA), fluorometry (Qubit, Invitrogen, Carlsbad, NM, USA) and quantitative PCR (PicoGreen dosage with Quant-iT™ PicoGreen® dsDNA Assay Kit, Invitrogen on ABI 7900HT, Applied Biosystems). After quality controls, six libraries (one per individual) were prepared using the TruSeq DNA Sample Prep Kits v2 and TruSeq Universal Adapters (Illumina, San Diego, CA, USA). A TruSeq Universal Adapter was used for each DNA library in order to separate reads from different individuals after DNA sequencing. Library sizes were checked on BioAnalyzer chips (Agilent Technologies, Santa Clara, CA, USA). Paired-end library sequencing was carried out on the Illumina MiSeq plateform (2 × 250 bp chemistry) at the GeT-PlaGe lab (GenoToul, Toulouse, France).

Genomic DNA was extracted from cell pellets using a QIAGEN QIAamp DNA mini kit and sheared using a Covaris Ultrasonicator (E220) to ~300 bp fragments. DNA concentration was measured using Qbit 4.0 reagents (ThermoFisher), and 200 ng of fragmented DNA was used for library preparation. End repair and A-tailing was carried out with NEBNext End repair reaction enzyme mix and buffer (E7442), and KAPA dual-indexed adapters (Roche) were ligated using the T4 DNA ligase kit from NEB (M0202). Post-ligation size selection was performed with AMPure XP beads (Beckman Coulter) before washing two times in 80% ethanol. Libraries were amplified using KAPA HiFi HotStart ready mix (Roche) and P5 and P7 primers (IDT). PCR program was as follows: 98 °C for 45 s, five cycles of 98 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. A further size selection and washing step was carried out after library amplification, and library quality was confirmed on Bioanalyzer chips (Agilent) and using a KAPA Library Quantification kit (Roche). Libraries were pooled and submitted for sequencing on NovaSeq 6000 at the New York Genome Center.