GSM9170391: HepG2, Active-seq, Rep3; Homo sapiens; OTHER
Genomic DNA was obtained from the ATCC (https://www.atcc.org/) Genomic DNA was sheared to an average of 180 bp in 8 microTube-50 AFA Fiber V2 Strips using a Covaris E220 evolution sonicator. 10 ng of input DNA was tagged using the Tagomics Active-Seq reaction kit and according to the manufacturer's instructions. DNA was mixed with the methyltransferase enzyme and tagging compound and incubated at 37 °C for 1 hour. The reaction was stopped using the reaction termination buffer, with an incubation at 55°C for 15 mins. Without purification, the sample was cooled to 10°C. To this, was added 9 µL of End Repair & A-Tailing Master Mix (3.5 µL KAPA End Repair & A-Tailing Buffer, 1.5 µL KAPA End Repair & A-Tailing Enzyme and 4 µL water). The mixture was mixed thoroughly by pipette aspiration and incubated at 20°C for 30 mins followed by a 65°C incubation for a further 30 mins. The sample was cooled to 10°C, and to it were added IDT xGen Stubby Adapters (2.5 µL of 15µM Stocks) and 22.5 µL of Ligation Master Mix were added (15 µL KAPA Ligation Buffer, 5 µL KAPA DNA Ligase and 2.5 µL water). The mixture was thoroughly mixed by aspiration then incubated at 20 °C for 30 minutes.Without purification, DNA mixed with Tagomics Active-seq capture compound and the mixture was incubated at 37 °C for 1 hour with shaking at 500 rpm. The DNA was purified from the reaction mixture using AMPure Beads (Beckman Coulter) (washing with 150 µL 80% Ethanol) with final elution in 30 µL PBST (10mM Phosphate Buffer, 1M NaCl, pH 7.5, 0.005 % Tween-20). 5 µL Dynabeads MyOne Streptavidin C1 beads (ThermoFisher) were washed with 150 µL of PBST. Capture-ready DNA was added to the beads (29 µL - 1 µL was taken for QC) and mixed with by aspiration until a homogenous mixture was achieved. The mixture was further incubated at 23 °C for 15 minutes, with shaking at 1000 rpm. Once completed, the beads were washed twice with 150 µL of PBST. Finally, the beads were resuspended in 20 µL of Tagomics Active-seq Release Buffer and incubated at 50 °C for 30 minutes at 1000 rpm. After release of the DNA, the beads were placed on the magnet and left for 3 minutes. The supernatant containing the unmodified DNA fraction was transferred to a sterile tube and can be stored at this point or used directly for amplification. A mixture of KAPA HiFi HotStart ReadyMix (2x, 25 µL), IDT Unique Dual Index Primer Pairs (5 µL of 10 µM Stock) and the enriched DNA library (20 µL) was prepared and subjected to amplification by PCR (up to 13 cycles). The amplified library was purified using 1X AMPure XP Beads (washed with 150 µL 80% ethanol) and eluted in 10mM Tris-HCl (pH 8.5), 0.01% Tween-20.