Journal: Protein & Cell
Article Title: SIRT7 antagonizes human stem cell aging as a heterochromatin stabilizer
Figure Lengend Snippet: SIRT7 is required for heterochromatin maintenance in hMSCs . (A) Schematic diagram showing the strategy for DamID-seq library construction. When the Dam-Emerin fused protein was expressed in hMSCs, the genomic DNA region near the nuclear envelope could be methylated by DNA adenine methyltransferase (Dam) at adenines. A parallel experiment “Dam only” was used to eliminate the background Dam signal. After genome extraction, the sequence containing methylated sites could be specifically cut by DpnI and amplified by PCR. The amplified fragments were then proceeded to DNA library construction and high-throughput sequencing. (B) Chromosome ideogram showing relative DamID signals in LADs across 23 chromosomes at MP (P6). The color key from blue to red shows low to high relative DamID levels, respectively. (C) Violin plot showing the DamID signal [log 2 (Dam-EMD/Dam)] in LADs in SIRT7 +/+ and SIRT7 −/− hMSCs at MP (P6). The white circles represent the median values, and the white lines represent the values within the IQR from smallest to largest. ***, P
Article Snippet: After DpnI digestion, adaptor ligation, DpnII digestion, PCR amplification and purification, the amplified DNA was sonicated and digested with AlwI (NEB) to remove the adaptors.
Techniques: Methylation, Sequencing, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing