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The genomic DNA was extracted based on the CTAB method. About 200 mg tissue after ground to powder by liquid nitrogen was transferred to a preheated (65℃) 2.0 mL tube with appropriate amount of CTAB lysis buffer and was mixed by vortex. The tube was incubated at 65℃ for 60 min, then was centrifuged at 10,000 rpm at RT for 5 min. The supernatant was extracted with one volume of phenol/chloroform/isopentanol (25:24:1) followed by centrifuging at 10,000 rpm at RT for 10 min in a new tube. 2/3 volume of precooled(-20℃) isopropanol was added into the tube and put at -20℃ for more than 2 hours to precipitate the DNAs, followed by centrifuging at 12,000 rpm for 15 min at RT. 75% ethanol was added to wash the pellet and removed by centrifuging, then the DNA pellet was air-dried for 3-5 min. The pellet was dissolved by 30-200 μL TE buffer for further study.After DNA extraction, 1 μg genomic DNA was randomly fragmented by Covaris, followed by fragments selection by Agencourt AMPure XP-Medium kit to an average size of 200-400 bp. Selected fragments were end repaired and 3’adenylated, then the adaptors were ligated to the ends of these 3’adenylated fragments. The products were amplified by PCR and purified by the Agencourt AMPure XP-Medium kit. The purified double stranded PCR products were heat denatured to single strand, and then circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library and qualified by QC. ssCir DNA molecule formed a DNA nanoball (DNB) containing more than 300 copies through rolling-cycle replication. The DNBs were loaded into the patterned nanoarray by using high density DNA nanochip technology. Finally, pair-end 150 bp reads were obtained by combinatorial Probe-Anchor Synthesis (cPAS). |
WGS |
GENOMIC |
RANDOM |
PAIRED
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