Illumina NovaSeq 6000 paired end sequencing; Exome-Seq of murine MB49 cell line - WT and gal-9-KO - in vitro
Exome-Seq of murine MB49 cell line - WT and gal-9-KO - in vitro
Cells were grown in DMEM supplemented with 5% fetal calf serum, 2mM glutamine and 5µg/mL gentamycin. Cell pellets were used for nucleid acid extraction. DNA was isolated using GeneJET Genomic DNA Purification kit (ThermoFisher). A total amount of 1.0μg genomic DNA per sample was used as input material for the DNA sample preparation. Sequencing libraries were generated using Agilent SureSelect All Exon kit (Agilent Technologies, CA, USA) following manufacturer's recommendations and x index codes were added to attribute sequences to each sample. Briefly, fragmentation was carried out by hydrodynamic shearing system (Covaris, Massachusetts, USA) to generate 180-280bp fragments. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and enzymes were removed. After adenylation of 3' ends of DNA fragments, adapter oligonucleotides were ligated. DNA fragments with ligated adapter molecules on both ends were selectively enriched in a PCR reaction. Captured libraries were enriched in a PCR reaction to add index tags to prepare for hybridization. Products were purified using AMPure XP system (Beckman Coulter, Beverly, USA) and quantified using the Agilent high sensitivity DNA assay on the Agilent Bioanalyzer 2100 system.