AB 3730 Genetic Analyzer paired end sequencing; Next generation sequencing for patients with hypomyelinating leukodystrophies
Next generation sequencing for patients with hypomyelinating leukodystrophies
Peripheral venous blood was collected from both patients and their families. Buffy coats of nucleated cells obtained from anticoagulated blood (ACD or EDTA) were resuspended in 15 ml polypropylene centrifugation tubes with 3 ml of nuclei lysis buffer (10 mM Tris-HCl, 400 mM NaCl and 2 mM Na2EDTA, pH 8.2). The cell lysates were digested overnight at 37°C with 0.2 ml of 10% SDS and 0.5 ml of a protease K solution (1 mg protease K in 1% SDS and 2 mM Na2EDTA). After digestion was complete, 1 ml of saturated NaCl (approximately 6M) was added to each tube and shaken vigorously for 15 seconds, followed by centrifugation at 2500 rpm for 15 minutes. The precipitated protein pellet was left at the bottom of the tube and the supernatant containing the DNA was transferred to another 15 ml polypropylene tube. Exactly 2 volumes of room temperature absolute ethanol was added and the tubes inverted several times until the DNA precipitated. The precipitated DNA strands were removed with a plastic spatula or pipette and transferred to a 1.5 ml microcentrifuge tube containing 100-200 pl TE buffer (10 mM Tris-HCl, 0.2 mM Na2EDTA, pH 7.5). The DNA was allowed to dissolve 2 hours at 37°C before quantitating. AmpliSeqTM ibrary was prepared following the modifiedIon AmpliSeqTM Library Preparation protocol (Pub No. MAN0006735).