MicroRNA roles in signaling during lactation: an insight from differential expression, time course and pathway analyses of deep sequence data
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1 MicroRNA roles in signaling during lactation: an insight from differential expression, time course and pathway analyses of deep sequence data Duy N. Do 1, 2, Ran Li 1, 3, Pier-Luc Dudemaine 1 and Eveline M. Ibeagha-Awemu 1* 1 Agriculture and Agri-Food Canada, Sherbrooke Research and Development Centre, 2000 College Street, Sherbrooke, Quebec, J1M 0C8, Canada 2 Department of Animal Science, McGill University, 21111, Lakeshore Road, Ste-Anne-de Bellevue, Quebec, J1M 0C8, Canada 3 College of Animal Science and Technology, Northwest A&F University, Xinong road 22, Shaanxi, , China * Corresponding author: Dr. Eveline Ibeagha-Awemu Agriculture and Agri-Food Canada Sherbrooke Research and Development Center, Sherbrooke, QC, Canada Eveline.ibeagha-awemu@agr.gc.ca
2 Supplementary figures Figure S1. The quality of RNA isolated from milk fat samples. The RIN (RNA integrity number) values ranged from 2.3 to 8.5. To see if the small RNA fraction was intact, we enriched the small RNA fraction using MiRVana kit which allows for the simultaneous extraction/enrichment of long RNA (>200bps) and small RNA (<200bp) fractions from the same sample. Analysis of RIN following enrichment showed an improvement in the RIN values of the long RNA fraction (B), while a sharp peak between nucleotide 25 and 200 showed that the small RNA fraction was intact (C). Furthermore, the RIN value of enriched RNA fractions (small and large) (B, C), depends on the quality of the starting material (A). As shown in figure, the RIN value of the starting total RNA was 7.9 (sample 42) while that of the long RNA fraction was 8.4 (E) and of the small RNA fraction, 2.4 (F). Since the small RNA fraction was intact, RNA used in library preparation in this study was not enriched. It has been shown that, some small RNA species are lost following the enrichment step (Podolska et al How the RNA isolation method can affect microrna microarray results. Acta Biochimica Polinica 58: ,
3 Figure S2. Daily milk yield and differences in milk yield between lactation days. (a) Daily raw milk yield (circles) and predicted lactation curve for milk yield using Wood s model (red curve). (b) Tukey's HSD (honest significant difference) test for effects of day on milk yield. a,b,c Means with different letters differ significantly at p<0.05.
4 Figure S3. Signalling pathways enriched for target genes of differentially expressed mirnas specific to each lactation transition stage. LAC: Lactogenesis; GAL: Galactopoiesis and INV: Involution
5 Supplementary Table Titles Table S1. Mapping statistics of mirna-seq reads Table S2. Length (nt) distribution of mirna reads Table S3. Know and novel mirna read counts TableS4. Summary of MiRDeep2 output for mirna prediction Table S5. Highly expressed mirnas during different stages of the bovine lactation curve and constituted 78.2, 84.2 and 81.4% of all read counts in LAC, GAL and INV stages, respectively. LAC: Lactogenesis; GAL: Galactopoiesis and INV: Involution TableS6. Pathways enriched for target genes of 15 abundantly expressed mirnas across lactation stages Table S7. Molecular and cellular functions (a), physiological system development functions (b) and pathways (c) enriched for target genes of mir-eia (a novel mirna) Table S8. Significantly differentially expressed mirnas between (a) galactopoiesis and lactogenesis, (b) involution and galactopoiesis, (c) involution and lactogenesis stages and (d) selected differentially expressed mirnas for downstream analyses Table S9. Pathways enriched for target genes of the most significantly differentially expressed mirnas at each lactation stage: a) mir-29b, b) mir-363, c) mir-874, d) mir-6524, e) mir-885 and f) mir- 2285t Table S10. Enriched (a) molecular and cellular functions and (b) physiology system development functions for target genes of differentially expressed mirnas for each lactation stage comparison Table S11. Enriched canonical pathways for target genes of differentially expressed mirnas for each lactation stage comparison Table S12. Enriched pathways for dynamic differentially expressed mirnas throughout the bovine lactation curve Table S13. Stage specific differently expressed mirnas. Table S13. Stage specific differently expressed mirnas Stage mirna basemean log2foldchange pvalue padj GAL bta-mir E E-05 GAL bta-mir E E-05 GAL bta-mir E GAL bta-mir E GAL bta-mir GAL bta-mir GAL bta-mir-106b GAL bta-mir GAL bta-mir-x GAL bta-mir-2285i GAL bta-mir-339b E-04 GAL bta-mir
6 GAL bta-mir GAL bta-mir GAL bta-mir-2285v GAL bta-mir-21-3p GAL bta-mir GAL bta-mir-190a GAL bta-mir-2284z GAL bta-mir-374a GAL bta-mir-345-5p GAL bta-mir-2284o GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir-x GAL bta-mir GAL bta-mir p GAL bta-mir GAL bta-mir-x GAL bta-mir GAL bta-mir-2285e GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir-20a GAL bta-mir GAL bta-mir GAL bta-mir GAL bta-mir-320a GAL bta-mir GAL bta-mir-30e-5p INV bta-mir-2284j E E-06 INV bta-mir E E-06 INV bta-mir E E-05 INV bta-mir E INV bta-let-7a-5p E INV bta-mir E INV bta-mir E E-04 INV bta-mir-30f E INV bta-mir p INV bta-mir p INV bta-mir-23b-3p INV bta-mir INV bta-mir INV bta-mir INV bta-mir INV bta-mir-331-3p INV bta-mir INV bta-mir-30b-3p
7 INV bta-mir INV bta-mir INV bta-let-7g INV bta-mir INV bta-mir INV bta-mir INV bta-mir INV bta-mir INV bta-mir INV bta-mir-x INV bta-mir INV bta-mir-345-3p INV bta-mir LAC bta-mir E E-11 LAC bta-mir-196a E E-08 LAC bta-mir-92b E E-07 LAC bta-mir-29d-5p E E-06 LAC bta-mir-2285ad E E-05 LAC bta-mir E LAC bta-mir E LAC bta-mir-380-3p E LAC bta-mir LAC bta-mir LAC bta-mir LAC bta-mir LAC bta-mir LAC bta-mir p LAC bta-mir LAC bta-mir LAC bta-mir p LAC bta-mir LAC bta-mir LAC bta-mir LAC bta-mir-181a LAC bta-mir LAC bta-mir LAC bta-let-7f LAC bta-mir LAC bta-mir p LAC bta-mir LAC bta-mir-2284a LAC: Lactogenesis GAL: Galactopoiesis INV: Involution Table S14. Enriched pathways for time dependent differentially expressed mirnas throughout the bovine lactation curve
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