Ez 96 dna methylation direct magprep kit

Only epithelial cells confirmed by transcriptome analysis were further selected for DNA library construction. We used 5 µL of protein lysis buffer containing 2.5 µL of M-digestion buffer (Zymo, Cat# D5044) and 0.5 µL of protease K (NEB, Cat# P8107S) to resuspend the bead-trapped nuclei. The genomic DNA in each cell was released after incubation at 50 °C for 1 h. Then, the genomic DNA lysates were stored at −80 °C, and we selected the genomic DNA of cells that were classified as epithelial cells through transcriptome analysis to perform DNA amplification. In brief, bisulfite conversion was carried out using the EZ-96 DNA Methylation-Direct™ Mag Prep Kit (Zymo, Cat# D5044). Specifically, we added only 32.5 µL of CT conversion reagent to 5 µL of single-cell genomic DNA lysate. We followed the steps of the single-cell whole-genome bisulfite sequencing workflow^{40 (link)} with minor modifications, including (1) We used random primers containing N6 sequences. (2) We performed a total of 4 rounds of olig1 tagging and skipped the Exo I digestion; instead, we removed the free primers by purification with 0.8 volume of Ampure XP beads (Beckman, Cat# A63882). Finally, we performed 16 cycles of the indexing PCR program at 98 °C for 15 s, 65 °C for 30 s, and 72 °C for 1 min. The DNA library for each cell was sequenced for 2 Gb (~0.6×) on the Illumina HiSeq 4000 platform.

Genomic DNA was extracted from FF tissue samples and FFPE tissue samples with AllPrep DNA/RNA Mini Kit (Qiagen, Germany, Catalog No. 80204) and AllPrep DNA/RNA FFPE Kit (Qiagen, Germany, Catalog No. 80234). Subsequently, the extracted DNA was quantified by the qubit dsDNA Customs Assay Facility (Thermal Fisher Science, USA, Catalog No.Q32851). The quality controlled criteria of CRC samples required that DNA content was more than 100 nanograms and the main band of agarose gel electrophoresis exceeded 500 bps. 50 nanograms of genomic DNA was taken from each sample, and EZ-96-DNA Methylation Direct MagPrep Kit (Zymo Research, USA, Catalog No. D5044) was used for bisulfite treatment of DNA.

Genomic DNAs were extracted from the FF specimens and FFPE tissue samples using the AllPrep DNA/RNA Mini Kit (Qiagen, Germany, Catalog No. 80204) and AllPrep DNA/RNA FFPE Kit (Qiagen, Germany, Catalog No. 80234) following the manufacturer’s instruction, respectively. Both genomic DNAs were quantified by the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA, Catalog No. Q32851). The quality control criteria of the EGC samples required that the DNA amount was greater than 100 ng and the main bands from the agarose gel electrophoresis were above 500 bp. Bisulfite treatment was implemented using 50 ng of genomic DNA of each FFPE tissue sample with the EZ-96-DNA Methylation-Direct MagPrep Kit (Zymo Research, USA, Catalog No. D5044) according to the manufacturer’s recommendations. Subsequently, a 50-marker EGC-LNM DNA methylation panel (Additional file 2: Table S2) was designed and used to characterize the methylation patterns in EGC-LNM patients with the EGC-LNM detection kit (AnchorDx, China, Catalog No. EGME-002). The methylation analysis by MethyLight approach was described earlier (details are in Additional file 2: Methods) [21 (link)]. The qPCR methylation analysis was performed on the Quant Studio 3 Real-Time PCR System (Thermo Fisher, USA). Then, the diagnostic model of LNM in EGC was established and validated based on methylation-specific qPCR data.