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DNA extraction from healthy or cancer samples was hold the Cell-Free DNA BCT (Strect, USA). We firstly gained the plasma from blood by centrifugation at 1900g for 10 min and 18000g for 10min at room temperature. Next, plasma sample was extracted using QIAamp Circulating Nucleic Acid Kit (55114, Qiagen) according to manufacturer’s recommendations. Approximately 4-5 ml of plasma could gain average 20-30 ng of Cell-free DNA. DNA was stored at -80? for further analysis. We used the DNA to build the methylation library. We mixed 3-30 ng Cell-free DNA and 1/1000-5/1000 ng Unmethylated Lambda DNA (D1521, Promega). Unmethylated Lambda DNA was sheared by a Covaris S220 instrument (Life Technologies, Thermo Fisher Scientific), and the size of fragments approximately were 200 bp. At first, DNA fragments were synthesized via end repair, adenylation of the 3? ends. The step we used reagent from NEBNext Ultra End Repair/dA-Tailing Module (E7442S/L, NEB). After mixture, reactions were incubated for 30 minutes at 20? and 65?. Secondly, the NEBNext Ultra Ligation Module (E7445S/L, NEB) reagent was used to ligate methylation adaptor. The process was incubated for 15 minutes at 20?. The adaptor sequence was Illumina next-generation sequencing. Thirdly, we used EZ DNA Methylation-Gold Kit™ (D5005, Zymo) to bisulfite-converted DNA according to manufacturer’s recommendations. Next, DNA was amplified by PCR which was carried out using primers. The primers came from Illumina next-generation sequencing. PCR was done as follows: 25?l KAPA HiFi HS Uracil +ready Mix (KK2801,KAPA)?20 ?l DNA ,10?M primer 1.0 and 10?M primer index, using the following two cycles: 45 s @98?p 10-14X of (15s @ 98?, 30 s @64?, 30 s @ 72?), 60 s @ 72?. Finally, we selected to effective captures (~290 bp) as the resulting library using Agencourt AMPure XP beads (Beckman Coulter). All binding and washing were done at the room temperature. Purity of the libraries of the concentrations were used Qubit dsDNA HS assay (Thermo Fisher) to check. The final DNA methylation libraries we sequenced using X-ten systems (Illumina). |
Bisulfite-Seq |
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unspecified |
PAIRED
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