title: GSM7967768: LM603; Homo sapiens; OTHER
library_construction_protocol: cells were resuspended in DNA Quick Extraction solution (Lucigen) to lyse the cells and extract genomic DNA. The cell lysate was incubated at 65 °C for 5 min, then 95 °C for 5 min, and stored at −20 °C. A 25 µl PCR-reaction contained 10 µl of genomic DNA, 0.5 µl 10 µM primer forward, 0.5 µl 10 µM primer reverse,  12.5 µl 2x GoTaq Long PCR Master Mix (Promega) and 1.5 µl H2O. The thermocycler setting consisted of one step at 95 °C for 1 min; followed by 18 cycles at 98°C for  10 s, 65°C for 15 s and 72°C for 15 s (wherein the annealing temperature was decreased by 0.5 °C per cycle); followed by 15 cycles at 98°C for 10 s, 58°C for 15 s and 72°C for 15 s; with one final step at 72°C for 5 min. The PCR products were cleaned up using AMPure beads according to the manufacturer's recommendations (Beckmann Coulter) and eluted in 50 µl 10 mM Tris. For barcoding, 2 µl of the purified DNA were added to 10 µl of 2x GoTaq Long PCR Master Mix, 2 µl of Nextera XT index 1 (i7) primer, 2 µl of Nextera XT index 2 (i5) primer (Illumina) and 4 µl of H2O. The PCR reactions were heated up to 95 °C for 3 min, followed by 8 cycles of 95°C for  30 s, 55°C for 30 s and 72°C for 30 s and a further elongation step for 5 min at 72°C. The second PCR products were again purified with AMPure beads and eluted in  27.5 µl 10 mM Tris. Cleaned-up PCR products were quantified using the SpectraMax Quant AccuBlue HighRange dsDNA Kit (Molecular Devices) on the SpectraMax 3x instrument (Molecular Devices). Equal amounts of DNA/sample were pooled and sequenced on an Illumina MiSeq instrument (Illumina) with a MiSeq Reagent Nano Kit v2 (500-cycles) (Illumina) according to the manufacturer's recommendations.