Supplemental Table S1. Primers used for relevant analysis. Gene Accession# Forward primer (5 -) Reverse primer (5 -) Application

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1 Supplemental Table S1. Primers used for relevant analysis. Gene Accession# Forward primer (5 -) Reverse primer (5 -) Application SCD1 NM_ CACGCAAAAGCAGGCTCAGGA GCATCTGGGCTCTCAGACACT RT-qPCR ACC1 NM_ GTTCTCATTGCCAACAATGGCA CAGCCAGCCCAAACTGCTTGCAC RT-qPCR FAS AF GCATCGCTGGCTACTCCTAC GTGTAGGCCATCACGAAGGT RT-qPCR LPL NM_ GGGTTTTGAGCAAGGGTACA GCCACAATGACCTTTCCAGT RT-qPCR CD36 X91503 GTACAGATGCAGCCTCATTTCC TGGACCTGCAAATATCAGAGGA RT-qPCR FABP1 FJ GTTCATCATCACCGCTGGCT CCACTGCCTTGATCTTCTCCC RT-qPCR DGAT1 NM_ CCACTGGGACCTGAGGTGTC GCATCACCACACACCAATTCA RT-qPCR DGAT2 NM_ CATGTACACATTCTGCACCGATT TGACCTCCTGCCACCTTTCT RT-qPCR PLIN2 NM_ TTTATGGCCTCATGCTTTTGC CTCAGAGCAGACCCCAATTCA RT-qPCR FGF21 XM_ AATATCACGGGTCAGGCGTC GGACTCACAGCTGACTGGAC RT-qPCR CPT1a NM_ TCGCGATGGACTTGCTGTATA CGGTCCAGTTTGCGTCTGTA RT-qPCR CPT2 NM_ TTTGGCATTGGGTACTCCGT TATGCTGGTGAAACAGAGGCT RT-qPCR ACOX1 NM_ ACCCAGACTTCCAGCATGAGA TTCCTCATCTTCTGCACCATGA RT-qPCR HMGCS2 NM_ GACATCGCAGTCTACCCCAG CATCGAGAGTGAAAGGCCGA RT-qPCR TLR2 NM_ GGCTGTGGAAGGCGCTGCAA GCCATCGCAGACACCAGTTG RT-qPCR TLR4 NM_ GGACCCTTGCGTACAGGTTG GGAAGCTGGAGAAGTTATGGC RT-qPCR TNF-a NM_ CTTCTGCCTGCTGCACTTCG GAGTTGATGTCGGCTACAACG RT-qPCR

2 IL-1a NM_ GGCCAAAGTCCCTGACCTCT CTGCCACCATCACCACATTC RT-qPCR IL-6 NM_ GGAGGAAAAGGACGGATGCT GGTCAGTGTTTGTGGCTGGA RT-qPCR SREBP1c NM_ CACTCGTCTTCCTCTGTCTC GAGTGACTGGTTCTCCATAG RT-qPCR SREBP2 NM_ AGAGCAAACTCCTGAAGGGC GGAGGCGACATCAGAAGGAC RT-qPCR SCAP NM_ CATCAAGCTCTACTCCATCC CAATGGCAGCGTTGTCCAGCA RT-qPCR INSIG1 NM_ ACTAAAGCCTGACTGCCAGC GATTGTCTGCGTAGCACCCT RT-qPCR PPARa FJ CATAACGCGATTCGTTTTGGA CGCGGTTTCGGAATCTTCT RT-qPCR LXRa NM_ CCCCATGACCGACTGATGTT TGTCCTTCATCTGGCTCCACC RT-qPCR NF- B (p65) NM_ TTTTTCACAAGCTGACGTGCAC GCTCTTGAAGGTCTCGTACGT RT-qPCR NF- B (p50) NM_ GTCAAACTCCAGAATGGCAGA GAAATCCTCTCTGTTTAGGTTGCTC RT-qPCR SFRS4 NM_ ATGGCAGTTACGGTTCTGGAC CCTGCCTGACGCATATAATCC RT-qPCR PA CTCCCACGGTGAACCAACTCT GCATCTGGGCTCTCAGACACT CHART and Methylation for area A PB GTGCCCATCCATTTGCGAATTG GGCTCGGCGCAATCTGCTGT CHART and Methylation for area B PS GTGCCCATCCATTTGCGAATTG GGTGCCACCTCGTCCTGCCGT ChIP The source files used to derive the primers are indicated (accession#).

3 Supplemental Table S2. Primer efficiency and qpcr performance for each gene. Gene Median Median relative mrna (Holstein) Ct 1 Ct 2 Slope 3 (R 2 ) 4 Efficiency 5 abundance 6 1/E Ct 7 % SCD FASN ACACA LPL CD FABP DGAT DGAT PLIN FGF CPT1A CPTII ACOX HMGCS

4 TLR TLR TNF IL1A IIL SREBP SREBP SCAP INSIG PPARA NR1H RELA NFKB SFRS SUM The median is calculated considering all cows. 2 The median of Ct is calculated as [Ct gene Ct internal control] for each cow. 3 Slope of the standard curve.

5 4 R 2 stands for the coefficient of determination of the standard curve. 5 Efficiency is calculated as [10 (-1 / Slope) -1]. 6 relative mrna abundance = 1/ Efficiency Median Ct 7 1/E Ct = relative mrna abundance/ relative mrna abundance

6 RNA extraction, PCR, and primer design and evaluation. Biopsy samples were powdered in a mortar under liquid nitrogen and was weighted (~50 mg) and immediately subjected to RNA extraction using ice-cold Trizol (Invitrogen Corp.). Genomic DNA was removed from RNA with DNase using RNeasy Mini Kit columns (Qiagen, Germany). RNA concentration was measured using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies). The purity of RNA (A260/A280) for all samples was above 1.9 as required. RNA quality was assessed by gel electrophoresis (shown in below). For cdna generation, 1.5 μg of total RNA were transcribed in reverse using SuperScript II (Invitrogen) and the general conditions as previously described (Goldammer et al., 2004). The cdna was purified with the High Pure Purification kit (Invitrogen) and subsequently diluted to a final effluent volume of 100 μl (15 μg/ul). For each measuring point, aliquots of 5 μl were supplemented with amplification primers (20 pm) and the components of the SYBR Premix Ex Taq TM kit (Takara Co., Otsu, Japan) for PCR reaction. Amplification cycles consisted of an initial denaturation (95 C, 10 min) followed by 40 cycles of annealing (60 C, 30 s), elongation (72 C, 1 min), fluorescence acquisition (83 C, 5 s) and melting (95 C, 30 s) on an ABI 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the recommendations in the instruction manual. Finally, the melting curves of the products were recorded. Quality of all PCR products was also visualized and validated in agarose gel electrophoresis. And qpcr performance for each gene were reflected by the series diluted standard curve and actual Ct value from each sample (shown in Suppl. Table 2). Design and evaluation of primers. Primer features for genes are shown in Suppl. Table 1. Primers were designed using Premier 6.0 software (Premier Biosoft International, USA). When possible, primers were designed to fall across exon exon junctions. Primers were aligned against publicly available databases using BLASTN at NCBI and bos taurus nucleotides were selected as database for potential unintended products. Prior to qpcr primers were tested in a 20 μl PCR reaction using the same protocol described for qpcr except for the final dissociation protocol. For primer testing we used mixed liver cdna (mixture from all 12 different bovine liver) to ensure

7 identification of desired genes. Only those primers that did not present primer-dimer, a single band at the expected size in the gel, and had the right amplification product (verified by sequencing) were used for qpcr. The accuracy of a primer pairs also was evaluated by the presence of a unique peak during the dissociation step at the end of qpcr. Reference Goldammer, T., H. Zerbe, A. Molenaar, H. J. Schuberth, R. M. Brunner, S. R. Kata, and H. M. Seyfert Mastitis increases mammary mrna abundance of betadefensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clinical and diagnostic laboratory immunology 11(1):

8 Supplemental Figure S1. Gel electrophoresis for RNA quality validation.

QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 2007)

QUANTITATIVE RT-PCR PROTOCOL (SYBR Green I) (Last Revised: April, 007) Please contact Center for Plant Genomics (CPG) facility manager Hailing Jin (hljin@iastate.edu) regarding questions or corrections.