Vα14 iNKT cells were isolated by flow cytometry and DNA was isolated using the PureLink genomic DNA mini kit (Life technologies). DNA was fragmented. 1.5 μg of the fragmented DNA was used for the library preparation and bisulfite treatment was done as described in the CMS-IP seq section. After the bisulfite conversion the purified DNA was amplified for 4 cycles (low amplification) using Kapa HiFi Uracil+ (Kapa Biosystems). 2 independent WGBS samples per genotype were evaluated.
In describing the WGBS data, we use the term “DNA modification” (5mC+5hmC) in preference to “DNA methylation” because bisulfite sequencing does not distinguish between 5mC and 5hmC54 (link), and because dot blot analysis shows persisting 5hmC in Tet2-Tet3 DKO thymocytes (Supplementary Fig. 1b ) that is most likely deposited by TET1 (Supplementary Fig. 1a ). However, the term “DNA methylation” is approximately correct, since 5hmC represents only a small percentage of total modified cytosines in T cells (<10% of 5mC)14 (link),55 (link).
To examine changes in DNA modification at the level of individual TSS regions and gene bodies, we calculated the average change in DNA modification in each gene body and at each promoter/TSS region (DMR discovery;Supplementary Methods ), and plotted the values against the change in expression of the corresponding gene (Supplementary Fig. 6b ).
In describing the WGBS data, we use the term “DNA modification” (5mC+5hmC) in preference to “DNA methylation” because bisulfite sequencing does not distinguish between 5mC and 5hmC54 (link), and because dot blot analysis shows persisting 5hmC in Tet2-Tet3 DKO thymocytes (
To examine changes in DNA modification at the level of individual TSS regions and gene bodies, we calculated the average change in DNA modification in each gene body and at each promoter/TSS region (DMR discovery;
