Table 2.

Multiplex nested PCR primer for DNA methylation analysis.

Gene	Primer Sequence (5′–3′)	Amplicon Length (bp)	Genomic Localization (EquCab3.0)	Number of CpGs in Inner Amplicon	Number of Sequenced CpGs
CYP19A1	Outer forward: TTTTAGTTTTGATTGGTTGTTTTT	317	1:140151317-140151633	-	-
	Outer reverse: CTAAACCCCATAAAACATCTCTTAC
	Inner forward: TTTTTTTTGTAAGATTAGTGAGTATATTTA	212	1:140151386-140151597	4	3
	Inner reverse: TTTCCAAAATTAAAAAACATAACC
DNMT1	Outer forward: AATTTTTTTTAAGAGTTTGGTATGG	264	7:51536766-51537029	-	-
	Outer reverse: ACCAATCCTCCTCTTTATACTAAAA
	Inner forward: GAGTTTGGTATGGTATATAAGTGTTGA	229	7:51536778-51537006	9	8
	Inner reverse: AAAAAACTAACCCTAAACTCACATC
DNMT3A	Outer forward: GGGATTGATTAGATTTTTTAGAGAAG	316	15:71656615-71656930	-	-
	Outer reverse: TAATAACACTAAATCCCTCCAAAAC
	Inner forward: TAGGAGTTTAGTGGGGGAATAGT	200	15:71656665-71656864	6	4
	Inner Reverse: ATAAAATAAATAAAACCCCTACACC
DNMT3B	Outer forward: TTAAAGGGGGAATAGTAGAAGTTTA	388	22:24214245-24214632	-	-
	Outer reverse: CAACTCCAAAAATATTTAAAATCAC
	Inner forward: TATAGAGGATGGATTTGGGATTTTA	240	22:24214309-24214548	10	8
	Inner reverse: ACTAAACACTCCCTACCCTAATACC
ESR1	Outer forward: TTGTGGTAGGTATGAATATTTATGTG	334	31:15363114-15363447	-	-
	Outer reverse: ATTACATATACAACCAACCACAAAC
	Inner forward: AATTTTTAGTGGGAGGAAGTATAGTAT	226	31:15363156-15363381	9	8
	Inner reverse: ACATAAACTAACAAAAAACATCCC
NANOG	Outer forward: TGGAAATATGGTGAATTTATAGGTAT	387	6:36542573-36542959	-	-
	Outer reverse: AACTTAAATATCCAAACAAAAAACC
	Inner forward: TTGGTAGATAGGATTAATTGAGAATT	237	6:36542585-36542821	8	7
	Inner reverse: CAAACAAAAAACCTTAAAAAAATAC
PTGES2	Forward: GATTTATTTAAGAGTGGGGGAGGT	205	25:32059289-32059493	17	11
	Reverse: CAATATAAAACCCCAACC
SOX2	Outer forward: ATTTTTAATATAGAATAAATTATGGAGAAG	302	19:22733114-22733417	-	-
	Outer reverse: AAATAAAAATAAAACAAAACAAAATAAATA
	Inner forward: ATAGAATAAATTATGGAGAAGTAAGGAG	253	19:22733122-22733376	19	13
	Inner reverse: CTATCCTACTAAAATTTCAAAAACC