DOI: 10.4231/8caa-ws64
title: Transcriptome analysis reveals disruption of circadian rhythms in late gestation dairy cows may increase risk for fatty liver and reduced mammary remodeling.

description: &lt;p&gt;The data here are the Supplemental Tables and Supplemental Figures of the manuscript (Under review).&lt;/p&gt;

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&lt;p&gt;Our previous studies found that circadian disruption of late gestation dairy cows, increased insulin resistance and decreased mammary development.  The aim of these studies were understand how circadian disruption impacted hepatic function and mammary development.  At 35 d before expected calving (BEC)  cows were assigned to either control (C) or phase-shifted treatments (PS), the C cows were exposed to 16 h light and 8 h of dark, whereas PS cows were light-dark cycle 6 h forward phase shifts every 3 d.  At 21 d BEC, liver and mammary were biopsied, and RNA was isolated  (n=6 CON and n=6 PS per tissue).  Libraries were prepared and sequenced on an Illumina Platform using paired end reads.  Data in the Tables published here describe these results.&lt;/p&gt;

&lt;p&gt;Supplemental Tables S1-S17&lt;/p&gt;

&lt;p&gt;1) Supplemental Table S1. Sample quality statistics.&lt;/p&gt;

&lt;p&gt;Describes quality of 24 samples (12 liver; 12 mammary-MG) of total RNA sequenced (RNA-seq) on an Illumina Platform using 150 bp, paired end reads.  Samples were isolated from liver and mammary biopsies taken from control (C) and phase-shifted (PS) treated multiparous dairy cows taken at 21 days before expected calving (BEC). &lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;
font-family:" times=""&gt;C was exposed to 16 h light and 8 h of dark. PS was exposed to 16 h light to 8 h dark, but phase of the light-dark cycle was shifted 6 h every 3 d.&lt;span style="mso-spacerun:yes"&gt;  &lt;/span&gt;At 21 d BEC, liver and mammary were biopsied. RNA was isolated&lt;span style="mso-spacerun:yes"&gt;  &lt;/span&gt;(n=6 CON and n=6 PS per tissue).&lt;/span&gt; Data include number of reads, number of bases, &lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;
font-family:" times=""&gt;and the mean Illumina quality score (Q-score) across samples was 35.7 &plusmn; 1.3.&lt;span style="mso-spacerun:yes"&gt;  &lt;/span&gt;The Q-score predicts the probability of a base calling error; the higher the score the more reliable the base call.&lt;span style="mso-spacerun:yes"&gt;  &lt;/span&gt;A base call with a quality score of Q40, predicts one base call in 10,000 is incorrect, whereas Q30 predicts one base call in 1,000 is incorrect. &lt;/span&gt;&lt;span new="" roman="" style="font-size:12.0pt;
line-height:107%;font-family:" times=""&gt;Data were deposited at NCBI&rsquo;s Gene Expression Omnibus (GEO accession &lt;/span&gt;&lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;
font-family:" times=""&gt;GSE168914&lt;/span&gt;&lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;font-family:" times=""&gt;)&lt;/span&gt;&lt;/p&gt;

&lt;p&gt;&lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;font-family:" times=""&gt;2) &lt;/span&gt;Supplemental Table S2. Total reads, mapped reads and percent mapped &lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;
font-family:" times=""&gt;to the ENSEMBL bovine genome averaged&lt;/span&gt;&lt;/p&gt;

&lt;p&gt;Describes quality of 24 samples (12 liver; 12 mammary-MG) of total RNA sequenced (RNA-seq) on an Illumina Platform using 150 bp, paired end reads.  Samples were isolated from liver and mammary biopsies taken from control (C) and phase-shifted (PS) treated multiparous dairy cows taken at 21 days before expected calving (BEC). C was exposed to 16 h light and 8 h of dark. PS was exposed to 16 h light to 8 h dark, but phase of the light-dark cycle was shifted 6 h every 3 d.  At 21 d BEC, liver and mammary were biopsied. RNA was isolated  (n=6 CON and n=6 PS per tissue).&lt;/p&gt;

&lt;p&gt;Data included number of reads, and percent of &lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;
font-family:" times=""&gt;trimmed reads that mapped to the &lt;i style="mso-bidi-font-style:normal"&gt;Bos taurus&lt;/i&gt; reference genome (ARS-UCD1.2) available on ENSEMBL using the STAR aligner v.2.5.2b.&lt;/span&gt;&lt;!--[if supportFields]&gt;&lt;span style='font-size:12.0pt;
line-height:107%;font-family:"Times New Roman",serif;mso-fareast-font-family:
"Times New Roman";color:black;mso-ansi-language:EN-US;mso-fareast-language:
EN-US;mso-bidi-language:AR-SA'&gt;&lt;span style='mso-element:field-end'&gt;&lt;/span&gt;&lt;/span&gt;&lt;![endif]--&gt;&lt;/p&gt;

&lt;p&gt;&lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;
font-family:" times=""&gt;3) &lt;/span&gt;Supplemental Table S3.  Normalized read counts of liver analysis by sample, base mean, log fold difference, standard error, t-statistic, nominal p-value, and adjusted p-value.&lt;/p&gt;

&lt;p&gt;4) Supplemental Table S4.  Normalized read counts of mammary analysis by sample, base mean, log fold difference, standard error, t-statistic, nominal p-value, and adjusted p-value.&lt;/p&gt;

&lt;p&gt;5) Supplemental Table S5. Functional annotation analysis of gene up regulated in liver of PS cattle relative to control.  Functional annotation analysis was done using &lt;span new="" roman="" style="font-size:12.0pt;line-height:
107%;font-family:" times=""&gt;Database for Annotation, Visualization, and Integrated Discovery [DAVID; version 6.8&lt;/span&gt;]&lt;/p&gt;

&lt;p&gt;6) Supplemental Table S6. Functional annotation analysis of gene down regulated in liver of PS cattle relative to control. Functional annotation analysis was done using Database for Annotation, Visualization, and Integrated Discovery [DAVID; version 6.8]&lt;/p&gt;

&lt;p&gt;7) Supplemental Table S7. Functional annotation analysis of gene up regulated in mammary of PS cattle relative to control. Functional annotation analysis was done using Database for Annotation, Visualization, and Integrated Discovery [DAVID; version 6.8]&lt;/p&gt;

&lt;p&gt;8) Supplemental Table S8. Functional annotation analysis of gene down regulated in mammary of PS cattle relative to control. Functional annotation analysis was done using Database for Annotation, Visualization, and Integrated Discovery [DAVID; version 6.8].&lt;/p&gt;

&lt;p&gt;9) Supplemental Table S9. Ingenuity Canonical Pathways enriched with genes differentially expressed in liver between CON and PS cows.&lt;/p&gt;

&lt;p&gt;10) Supplemental Table S10. Ingenuity Canonical Pathways enriched with genes differentialy expressed in mammary between CON and PS cows.&lt;/p&gt;

&lt;p&gt;11) Supplemental Table S11.  Ingenuity Pathway Analysis prediction of upstream regulators of genes significantly (P&lt;0.05) affected by PS in liver tissue.&lt;/p&gt;

&lt;p&gt;12) Supplemental Table S12.  Ingenuity Pathway Analysis prediction of upstream regulators of genes significantly (P&lt;0.05) affected by PS in mammary tissue.&lt;/p&gt;

&lt;p&gt;13) Supplemental Table S13. Correlation coeffecient of gene counts in liver significantly (P&lt;0.05) related to area under the curve of isulin in each cow in response to intravenous glucose challenge test at 14 d BEC.&lt;/p&gt;

&lt;p&gt;14) Supplemental Table S14. Functional annotation analysis of normalized gene read counts in liver that correlated (P&lt;0.05) with insulin area under the curve at 14 d BEC.&lt;/p&gt;

&lt;p&gt;15) Supplemental Table S15. Correlation coeffecient of gene counts in mammary significantly (P&lt;0.05) related to area under the curve of isulin in each cow in response to intravenous glucose challenge test at 14 d BEC.&lt;/p&gt;

&lt;p&gt;16) Supplemental Table S16. Functional annotation analysis of normalized gene read counts in mammary that correlated (P&lt;0.05) with insulin area under the curve at 14 d BEC.&lt;/p&gt;

&lt;p&gt;17) Supplemental Table S17. Genes differentially expressed in mammary tissue between PS and control cattle that overlap with genes altered in mammary epithelial cells by conditional knockout of insulin receptor in late pregnant mice (pregnnacy day 14.5)1 or overlap with genes identified as transcriptional targets of BMAL1 in the mouse mammary epithelial cell line HC11 using ChIP-seq analysis.&lt;/p&gt;

&lt;p&gt;&lt;b style="mso-bidi-font-weight:normal"&gt;&lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;font-family:" times=""&gt;Supplemental Figure S1&lt;/span&gt;&lt;/b&gt;&lt;span new="" roman="" style="font-size:12.0pt;line-height:107%;font-family:" times=""&gt;. The relationship between hepatic expression levels of EEF2, CARMIL3, BMPR2, and FERMT2&lt;span style="mso-spacerun:yes"&gt;  &lt;/span&gt;(read counts, log base 2) at 21 d before expected calving with insulin area under the curve (AUC) in response to intravenous glucose tolerance test at 2 weeks before expected calving in cattle exposed to control light-dark cycles (blue triangles) and 6 h light-dark phase-shifts (black dots) every 3 days.&lt;span style="mso-spacerun:yes"&gt;  &lt;/span&gt;&lt;/span&gt;&lt;/p&gt;

&lt;p&gt;&lt;strong&gt;Supplemental Figure S2&lt;/strong&gt;. The relationship between mammary expression levels of LAMP2, PLAU, ITGB4, and ZNF445  (read counts, log base 2) at 21 d before expected calving with insulin area under the curve (AUC) in response to intravenous glucose tolerance test at 2 weeks before expected calving in cattle exposed to control light-dark cycles (blue triangles) and 6 h light-dark phase-shifts (black dots) every 3 days.&lt;/p&gt;

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description: Circadian disruption increased insulin resistance and decreased mammary development in late gestation cows. The effect of circadian disruption on liver and mammary transcriptomes was measured to understand how it impacted hepatic function and...