GSM6195487: AphPulsed-MLH1KO-clone1-WGS; Homo sapiens; OTHER
Approximately 20 million cells were harvested by centrifuging at 500 g for 5 min at 4 degree. High molecular weight DNA was prepared using Nanobind Big DNA CBB kit (Circulomics) according to manufacturer's instructions. In brief, cells were washed with PBS and resuspended in 20 ul PBS followed by 20 μl proteinase K and 20 μl CLE3 buffer. The tube was pulse vortexed 10 times and incubated in thermomixture at 55 degree for 10 minutes at 900 rpm, followed by RNAse A treatment for 3 minutes at room temperature (RT). To lyse the cells, 200 μl of BL3 buffer were added, pulse vortexed 10 times, and incubated at 55 dgree at 900 rpm for 10 minutes. Nanodisk was added to the cell lysate followed by 300 μl of isopropanol, mixed 5x by inversion, followed by further mixing at 9 rpm using tube rotator at RT for 10 minutes. Tubes were placed on magnetic tube rack in a way that Nanobind bead remained captured near the top to avoid touching DNA with pipette tip. Supernatant were discarded and beads were washed 4 times each with buffer CW1 and CW2. High molecular weight DNA was eluted by 100 μl of Qiagen EB buffer. Purity of the DNA was analyzed using TapeStation. DNA samples were sheared 10-13 kb fragments for library preparation. WGS libraries of the samples were sequenced on PacBio SMRT cell using Sequel II with standard PacBio protocol. Samples yielded 1.78 M to 2.35 M ccs reads, with mean read length 9.44 kb and mean quality QV38.0.