GSM7103838: S_AJ_ALT_pos18; Homo sapiens; OTHER
gDNA was obtained from Coriell DNA DSB ends of nuclease-digested gDNA were repaired and 3' adenylated using the NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB E7546) as described by the manufacturer with the following modification: Reaction volume was halved by adding half volume of the reagents. Labeling of DSB ends with BreakTag linker ligation was performed using the NEBNext® Ultra™ II Ligation Module (NEB E7595) according to manufacturer's recommendation with the following modifications: Reaction volume was halved by adding half volume of reagents, and USER enzyme digestion step was omitted. BreakTag linker was used at a final concentration of 50 nM per sample. Labeled DNA was size-selected using DNA AMpure XP beads (0.7x volumes) in order to remove unligated linkers and eluted in nuclease-free H2O. Tagmentation with in-house Tn5 was performed in 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 25% dimethylformamide (DMF, Sigma Aldrich 227056). Tagmentation reactions were assembled using 100-200 ng of DNA as input. Hyperactive Tn5 was used at a final concentration of 1.25 ng/µL per reaction. The tagmentation mix was then incubated at 55ᵒC for 5 minutes in a preheated thermocycler followed by termination with 0.2% SDS at room temperature for 5 minutes. Libraries were amplified with the NEBNext® Ultra™ II Q5® Master Mix (M0544). Amplified and barcoded samples were size-selected in order to remove PCR byproducts by performing two consecutive 0.5x volume right-tail + 0.35x volume left-tail size selection using DNA AMpure XP beads. Final libraries were quantified using Qubit dsDNA High Sensitivity Assay kit and fragment size distribution was assessed in a BioAnalyzer High Sensitivity DNA chip