GSM8331727: HAPE patient 010 (RRBS); Homo sapiens; Bisulfite-Seq
Use a DNA extraction kit to extract DNA, use agarose gel electrophoresis to analyze the degree of DNA degradation and whether there is RNA contamination, and use Qubit to accurately quantify the DNA concentration. After the sample passes the testing, it is first digested with the methylation-insensitive restriction enzyme, MspI; The digested DNA fragments undergo end repair, A-tailing, and ligation with sequencing adapters that have all cytosines methylated; The gel is cut to select DNA fragments between 150-300bp in length; Bisulfite treatment is then carried out using the EZ DNA Methylation Gold Kit (Zymo Research). Through this treatment, unmethylated Cs turn into U (becoming T after PCR amplification), while methylated Cs remain unchanged. Finally, PCR amplification is conducted to generate the final DNA library. After the library construction is completed, Qubit 2.0 is used for preliminary quantification, diluting the library to 1ng/µ l. Then, Agilent 2100 is used to detect the length of the inserted fragments in the library. After meeting expectations, Q-PCR is used to accurately quantify the effective concentration of the library (effective concentration>2nM) to ensure the quality of the library.