5336 Nucleic Acids Research, 2009, Vol. 37, No. 16 Figure 3. DNA methylation of Ly49d and Ly49r 50-region in the F1 hybrid of 129/S6 and B6. (A) Ly49D/R expression proﬁle of 129S6xC57Bl/6 F1 hybrid fresh spleen NK cells (DX5+, CD3e ). The Ly49D/R-expressing (12A8+, A1 ) population (gated) and non- expressing (12A8 , A1 ) were sorted and analysed for DNA methyla- tion. (B) Sodium bisulﬁte sequencing of 12A8-positive/A1-negative NK cells (Ly49D/R positive) and 12A8-negative NK cells (Ly49D/R nega- tive). Ly49d and r diﬀer in the position of one CpG dinucleotide but also have other polymorphisms in this region ( 700 bp). All clones presented here are unique. There are no antibodies available that bind Ly49D but not Ly49R or vice-versa, making receptor/allele-speciﬁc sorting impossible because the 4E5 and 12A8 antibodies (14) detect both receptors. However, the 4E5 antibody also detects Ly49O129 and Ly49V129 where as 12A8 binds to Ly49AB6 as well (36). The percentage of NK cells expressing Ly49D (stained with 4E5) and R (stained with 12A8 antibody) in B6 and 129S6 strains respectively is 50-60% (data not shown). In order to speciﬁcally sort Ly49D and R expressing cells in 129S6/B6 F1 hybrid, splenic lymphocytes were co-stained with 12A8 (anti- Ly49D, R and AB6) and A1 (anti-Ly49AB6) antibodies. The A1 antibody also detects Ly49P129 and V129 with low binding aﬃnity (36). Assuming Ly49d and r are allelic, we hypothesized that, based on the lack of a bimodal DNA methylation pattern observed for Ly49D and H expressing NK cells in B6, both Ly49d and r 50-regions should be hypo-methylated. We FACS sorted 12A8-positive/A1-negative NK cells (Figure 3A). This population should include Ly49D- single positive, Ly49R single-positive and Ly49D and R double positive cells. A few polymorphisms in the 50-region of Ly49d and r, assayed by sodium bisulﬁte sequencing, allow for their distinction (Figure 3B). Figure 4. Detection of Ly49dB6 and Ly49dNOD cDNA by single-cell RT–PCR and Southern blot. Ly49dB6-speciﬁc and Ly49dNOD-speciﬁc probes were hybridized to identical blots of ampliﬁed cDNA generated from single-cell RT–PCR on FACS sorted F1 hybrid splenic NK cells. This ﬁgure shows a representative experiment (one set of PCRs) where seven individual cells show one or more products. Three cells contain Ly49dB6 only, two cells contain Ly49dNOD only and two cells contain both products. C indicates a lane with cDNA from JEG-3 carrier cells only (no sorted NK cell) and N indicates the no-template control lane. After ﬁve sets of independent PCRs 4 of 9 single cells in this combination of F1 cells showed bi-allelic expression for Ly49d (data not shown). We also sorted 12A8 negative (Ly49D/R non- expressing) NK cells and analyzed the 50-region of Ly49d and r for this population as well (Figure 3B). As expected, the 50-region of Ly49d and r in the Ly49D/ R-negative NK cells was hyper-methylated for all the sequenced clones. With the exception of two clones for Ly49d and one clone for Ly49r, all other clones (16 clones) are hypo-methylated in Ly49D/R-positive NK cells. The most 50 CpG of all of the nine independent Ly49R-positive clones is methylated in Ly49D/R-positive NK cells. However, the rest of the methylation pattern does not resemble that of the Ly49D/R-negative cells. We also sequenced the Ly49r Pro2 region from Ly49R- postive NK cells of the 129/SvEvTac mouse (the parent of the F1 hybrid) to gauge the methylation of the most 50 CpG. We observed the same methylation pattern as in the F1 (Supplementary Figure 1) possibly indicating that this pattern of methylation is speciﬁc to the Ly49r locus. The bimodal DNA methylation observed for the inhibitory Ly49a and c is not observed for Ly49d and r, rather the pattern of DNA methylation resembles that observed for Ly49h. Detection of bi-allelic expression of Ly49d The recent sequencing and assembly of the NOD Ly49 cluster (7) provided an opportunity to test allelic expres- sion of activating Ly49 receptors. While there may be some question as to the allelic relationship of Ly49dB6 and Ly49r129, the NOD strain has a deﬁnite Ly49d allele with 98.5% identity to the B6 Ly49d sequence at the level of cDNA (7,38). In order to directly test the quality and quantity of allelic expression of Ly49D, we single-cell FACS sorted NK cells (NKp46+, DX5+, NK1.1+, CD3e ) from B6/NOD F1 hybrid spleen and performed single-cell RT–PCR (27) followed by Southern blot and hybridization with allele-speciﬁc probes (see Figure 4 for example). We assayed 88 NK cells and detected cDNA for Ly49dB6 and/or Ly49dNOD in 40 cells where cDNA from both Ly49dB6 and Ly49dNOD was detected in 22 of these 40 cells indicating 55% bi-allelic expression (Table 2). We never detected any products from wells that only