Single-Cell Whole-Genome Bisulfite Sequencing

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.