Nucleic Acids Research, 2009, Vol. 37, No. 16 5333 Table 1. Oligonucleotide sequences of primers and probes Ly49h forward ﬂanking bisulﬁte primer 50-ATA GGG GAA TGT TAG GGT TAA AAA G-30 Ly49h reverse ﬂanking bisulﬁte primer 50-ATT TAA CCT AAT ATA ACA CAA CCA A-30 Ly49h forward nested bisulﬁte primer 50-GGA TAT ATG TTT TGT TTT TTT TGG T-30 Ly49h reverse nested bisulﬁte primer 50-TAA CAC AAC CAA AAA AAC TCT CAA C-30 Ly49d forward ﬂanking bisulﬁte primer 50-TAT TAA GAT GTA ATT AGT ATG ATT TAA T-30 Ly49d reverse ﬂanking bisulﬁte primer 50-ACA ATA CAT TTA TAC ACT TCA CCT AA-30 Ly49d reverse nested bisulﬁte primer 50-CCA AAT ACT ACA AAA AAA ATA ACT ATA T-30 Ly49d forward nested bisulﬁte primer 50-AGG TAG AGT TAT AGG TAA TAA TAG T-30 Ly49d/r reverse nested bisulﬁte primer 50-TTC CTC TAC CTT AAT TTC TTA AC-30 Ly49d Exon 2 forward primer 50-CGG AAG CCT GAA AAA GCT CG-30 Ly49d Exon 4 reverse primer 50-TCA CAC AGT ATG TTT TGA TCC C-30 Ly49h Exon 2 forward primer 50-GAA CAG CCA GGT GAG ACT T-30 Ly49h Exon 3-4 reverse primer 50-TGT TTG TGA CAA AGT TTT TTC AGT-30 Nkg2d Exon 2 forward primer 50-ACT ACC AGT CAA CCT GGA GAA-30 Nkg2d Exon 6 reverse primer 50-GAC ATA TCC AGT TGT TAG GGC AT-30 Ly49dB6/NOD forward Exon 2 primer 50-GCT GTG AGA TTC CAT AAG TCT TC-30 Ly49dB6/NOD reverse Exon 4 primer 50-GAT GCT GCA GTT ATT GTG GTG-30 Ly49dB6-speciﬁc oligonucleotide probe 50-CGG AAG CCT GAA AAA GCT CG-30 Ly49dNOD-speciﬁc oligonucleotide probe 50-AGC CTC GAA AAG CTG GCC TCA-30 Nkg2d-speciﬁc oligonucleotide probe 50-CAA TTC GAT TCA CCC TTA ACA CAT T-30 Ly49h Exon 2 inner reverse primer 50-AAA GTG ACC TCC TGC TCA CT-30 Ly49d forward EMSA probe 50-AGA AAA GGC CCA CAT TAC CCC AAC AGG GAC ATC CAT TCC TTC TAC-30 Ly49h forward EMSA probe 50-AGT AAA GGC CCA CAT TAC CCC AAT TGA GGC ATC CAT TCT TTC TAC-30 Ly49a forward EMSA probe (Plus two T-10-mers) 50-tttttttttt AGA AAA AGC CAA CTT TTT CCT CCA C tttttttttt-30 Ly49c forward EMSA probe (Plus two T-4-mers) 50-tttt AGA AAA CGC CAA CGT TTC AGA CAA ATT TTC CCT CCA C tttt-30 performed on 2 ml of the ﬂanking PCR product with the forward nested Ly49d primer (which is identical to Ly49r sequence) and Ly49d/r reverse nested bisulﬁte primer. After initial denaturation for 7 min at 958C, 35 cycles were performed, each consisting of 90 s at 958C, 55 s at 50 + 0.18C/cycle and 45 s at 728C with a ﬁnal extension of 7 min at 728C. The PCR products were electrophoresed on 1% agarose gels and correct size bands were extracted using the MinElute gel extraction kit (Qiagen). The pur- iﬁed products were cloned into the T-vector using the pGEMT-vector kit (Promega). Sequencing was performed using the SP6 primer by McGill University and Genome Que´ bec Innovation Centre sequencing facility. All clones included in the ﬁgures are unique as per criteria stated before (22). Combined bisulfite and restriction enzyme analysis For COBRA, gel puriﬁed fragments of Ly49d upstream and downstream regions were digested with TaqaI and BmgBI restriction enzyme (NEB), respectively to distin- guish between methylated (CpG) and unmethylated (TpG). Only fragments that were originally methylated in the gDNA and therefore not converted by sodium bisul- ﬁte treatment are cut. Gel puriﬁed Ly49h fragments were digested with TaqaI (NEB) and BsaAI (NEB) restriction enzymes. RNA extraction, RT–PCR and 5’ amplification of cDNA ends cDNA was generated from total B6 and NOD/ ShiLtJ spleens and FACS sorted B6 T-cell RNA per SuperScript III protocol. PCR was performed on the gen- erated cDNAs using primers speciﬁc for Ly49d, g, h, Nkg2d and actin. Ly49g and actin primers have been described before (22). All RT–PCR primer sequences are shown in Table 1. 50-Rapid ampliﬁcation of cDNA end (50-RACE) was performed on B6 spleen RNA using the FirstChoice RML-RACE kit (Ambion) per manufacturer’s protocol with Ly49h-speciﬁc primers. For the outer PCR, the Ly49h Exon 3–4 reverse primer used for RT–PCR was used as the outer 50-RACE primer in combination with the kit’s outer forward primer. For the inner PCR, Ly49h Exon 2 inner reverse primer in combination with the kit’s inner forward primer. The products were cloned into the T-vector using the pGEMT-vector kit (Promega). Sequencing was performed using the T7 primer by McGill University and Genome Qu e´bec Innovation Centre sequencing facility. Electrophoretic mobility shift assay Nuclear extract from FACS sorted B6 T-cells and IL- 2-expanded FACS-sorted B6 NK cells were prepared as described before (26). Electrophoretic mobility shift assay (EMSA) was performed with the following double- stranded probes located downstream of the transcriptional start site within exon1 of various Ly49 genes (only the forward strand is shown in Table 1): to control for length diﬀerences among the probes, poly-T tails were added to the shorter Ly49a and c probes. The YY1 anti- body (sc-281) was purchased from Santa Cruz Biotechnology and supershift experiments were performed as described before (26). Single-cell RT–PCR Single-cell RT–PCR and Southern blotting was performed as described before (27) with minor modiﬁcations. RNA from JEG-3, a human choriocarcinoma cell line (28), was used as carrier. Ly49d forward exon 2 and reverse exon 4 primers were used to speciﬁcally amplify Ly49d cDNA of both B6 and NOD/ShiLtJ. Ly49dB6-speciﬁc oligo probe and Ly49dNOD-speciﬁc oligo probe were used to