Nanopore sequencing of HEK-293T, 10 minutes sequencing, with selective sequencing , run 1
Qiagens QIAamp DNA mini kit was used to extract DNA from HEK-293T cells. Next, 2.5 x 106 or 1 x 106 cells were centrifuged at 1,400 x g for 5 minutes, and the resulting pellet was resuspended in 200 l PBS. DNA was then extracted according to the manufacturers protocol and eluted in 200 l H2O. The DNA concentration was measured using the RNA High Sensitivity assay on a Qubit fluorometer (Thermo Fisher Scientific, USA). DNA purity was assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Thermo Scientific, USA), to ensure OD 260/280 and OD 263/230 > 1.8. Approximately 400 ng of purified DNA in a total volume of 7.5 l in a 0.2 ml PCR tube was used as input for sequencing library preparation using Oxford Nanopore Technologies Rapid Sequencing kit (SQK-RAD004, version RSE_9046_v1_revB_17Nov2017) according to the manufacturers instructions. For fragmentation and transposase adapter attachment, 2.5 l FRA was added to the DNA and mixed by inversion. The sample was then incubated at 30C for 1 minute, followed by 80C for 1 minute, and finally cooled on ice. Sequencing adapters were then attached by adding 1 l RAP to the mixture and mixing by inversion. The sample with sequencing adapters was incubated at room temperature for 5 minutes, and then stored on ice until it was ready for sequencing.