Illumina HiSeq 2000 sequencing; Functional genomics of cattle through integration of multi-omics data
Cell sample collections. Skeletal muscle and subcutaneous fat samples were collected from Angus-crossbred steers slaughtered at the Virginia Tech Meat Center. Satellite cells were isolated from skeletal muscle by pronase digestion as described before (Leng et al. 2019). The isolated satellite cells were activated to proliferate as myoblasts by culturing in growth medium composed of Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 1% antibiotics-antimycotics. To induce myoblasts to differentiate into myocytes, myoblasts cultured in growth medium were switched to differentiation medium composed of DMEM and 2% horse serum for 2 days. Preadipocytes from subcutaneous fat were isolated by collagenase digestion as previously described (Hausman et al. 2008). To induce preadipocytes to differentiate into adipocytes, preadipocytes were initially cultured in growth medium (DMEM/F12, 10% FBS, 1% antibiotics-antimycotics) to reach confluency, then in induction medium (DMEM/F12, 10% FBS, 1% antibiotics-antimycotics, 10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine, and 200 μM indomethacin) for 2 days, and lastly in maintenance medium (DMEM/F12, 10% FBS, 1% antibiotics-antimycotics, 1 μg/mL insulin) for 10 days.Adult tissue collections. Procedures for tissue collection followed the Animal Care and Use protocol (#18464) approved by the Institutional Animal Care and Use Committee (IACUC), University of California, Davis (UCD).  Four cattle (2 males and 2 females) were slaughtered at UCD using captive bolt under USDA inspection at 14 months old and were intact male and female Line 1 Herefords that had the same sire, provided by Fort Keogh Livestock and Range Research Lab (Tixier-Boichard et al. 2021). Tissue samples were flash frozen in liquid nitrogen then stored at –80 °C until further assay processing.  Fetal tissue collections. Fetal sample collection and tissue collection were approved by the Institutional Animal Care and Use Committee (IACUC), University of Idaho (2017-67).  Four pregnant females Line 1 Herefords were slaughtered at UI meats lab using captive bolt under USDA inspection. Animals were provided by Fort Keogh Livestock and Range Research Lab (Tixier-Boichard et al. 2021). Tissue samples were flash frozen in liquid nitrogen then stored at –80 °C until further assay processing. Cell sample collections. Skeletal muscle and subcutaneous fat samples were collected from Angus-crossbred steers slaughtered at the Virginia Tech Meat Center. Satellite cells were isolated from skeletal muscle by pronase digestion as described before (Leng et al. 2019). The isolated satellite cells were activated to proliferate as myoblasts by culturing in growth medium composed of Dulbecco's Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 1% antibiotics-antimycotics. To induce myoblasts to differentiate into myocytes, myoblasts cultured in growth medium were switched to differentiation medium composed of DMEM and 2% horse serum for 2 days. Preadipocytes from subcutaneous fat were isolated by collagenase digestion as previously described (Hausman et al. 2008). To induce preadipocytes to differentiate into adipocytes, preadipocytes were initially cultured in growth medium (DMEM/F12, 10% FBS, 1% antibiotics-antimycotics) to reach confluency, then in induction medium (DMEM/F12, 10% FBS, 1% antibiotics-antimycotics, 10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM isobutyl methylxanthine, and 200 μM indomethacin) for 2 days, and lastly in maintenance medium (DMEM/F12, 10% FBS, 1% antibiotics-antimycotics, 1 μg/mL insulin) for 10 days. Adult tissue collections. Procedures for tissue collection followed the Animal Care and Use protocol (#18464) approved by the Institutional Animal Care and Use Committee (IACUC), University of California, Davis (UCD).  Four cattle (2 males and 2 females) were slaughtered at UCD using captive bolt under USDA inspection at 14 months old and were intact male and female Line 1 Herefords that had the same sire, provided by Fort Keogh Livestock and Range Research Lab (Tixier-Boichard et al. 2021). Tissue samples were flash frozen in liquid nitrogen then stored at –80 °C until further assay processing.   Fetal tissue collections. Fetal sample collection and tissue collection were approved by the Institutional Animal Care and Use Committee (IACUC), University of Idaho (2017-67).  Four pregnant females Line 1 Herefords were slaughtered at UI meats lab using captive bolt under USDA inspection. Animals were provided by Fort Keogh Livestock and Range Research Lab (Tixier-Boichard et al. 2021). Tissue samples were flash frozen in liquid nitrogen then stored at –80 °C until further assay processing. RNA-seq library construction. Tissue samples (Additional file18) were collected from storage at -80oC and ground to a powder using a mortar and pestle and liquid nitrogen. The tissue was next homogenized in QIAzol Lysis Reagent (Qiagen Catalog No. 79306) using a QIAshredder spin column (Qiagen Catalog No. 79656). After centrifugation, the lysate was mixed with chloroform, shaken vigorously for 15 sec, incubated for 2 – 3 min at room temperature, and centrifuged for 15 min at 12,000 x g at 4°C. The upper, aqueous phase was transferred to a new collection tube and 1.5 vol of 100% ethanol was added and mixed thoroughly by pipetting up and down several times. Total RNA was then isolated from the sample using the RNeasy Mini Kit (Qiagen Catalog No. 74106) according to the manufacturer's instructions. Contaminating DNA was removed by treating total RNA with DNase (AM1906, Ambion). Total RNA quantity was measured with the Quant-It RiboGreen RNA Assay Kit (Life Technologies Corp., Carlsbad, CA) and quality assessed by fragment analysis (Advance Analytical Technologies, Inc., Ankeny IA). Mammary gland RNA-seq and miRNA-seq library construction. The 16 animals used in this study were Holstein-Friesian Heifers from a single herd managed at the AgResearch Research Station in Ruakura, NZ. All experimental protocols were approved by the AgResearch, NZ, ethics committee, and carried out according to their guidelines. Samples were collected from the same animals at 5 time points: virgin state before pregnancy between 13 and 15 months of age (virgin), mid pregnant at 100 day of pregnancy, late pregnant ~2 weeks pre-calving, early lactation ~2 weeks post-calving, and at peak lactation, 34-38 days post-calving. Tissue samples were obtained by mammary biopsy using the Farr method (Farr et al. 1996). Lactating cows were milked before biopsy and sampled within 5 hours of milking. Biopsy sites were clipped and given aseptic skin preparation (povidone iodine base scrub and iodine tincture) and subcutaneous local anesthetic (4 ml per biopsy site). Core biopsies were taken using a powered sampling cannula (4.5 mm internal diameter) inserted into a 2 cm incision. The resulting samples of mammary gland parenchyma measured 70 mm in length, with a 4 mm diameter. Small slices from each sample were preserved for histology before mammary epithelial organoids were separated from surrounding adipose and connective tissue to allow for secretory-specific signals in the RNA-seq analysis. In preparation for isolating organoids, tissue samples were digested in a freshly prepared collagenase solution containing 0.2% collagenase A (Roche), 0.05% trypsin (1:250 powder, 100U/ml Gibco), hyaluronidase (Sigma), 5% fetal calf serum (Hyclone), Pen/Strep/Fungizone solution (Hyclone) or 5 µg/ml Gentamycin (Sigma) in DMEM/F12 (Gibco) with 10 ng/ml insulin. Samples were minced to a fine slurry and incubated in this freshly prepared collagenase solution (10 ml solution / g tissue) for 3.5 hours at 37°C in a 50 ml conical tube with slow shaking (120 rpm). Digested tissue was centrifuged at 453 xg for 10 min at 4°C, after which the supernatant and fat layers were discarded, and the pellet was gently resuspended in 5 ml DMEM/F12 without serum. A further 5 ml DMEM/F12 without serum was added, and the sample was centrifuged at 453 xg for 10 min at 4°C. The media was discarded, and the pellet was gently resuspended in 10 ml DMEM/F12 and centrifuged for another 10 min at 453 xg and 4°C. The media was discarded, and pellet resuspended in 10 ml DMEM/F12 for a third time, and the sample centrifuged in a series of brief spins achieved by allowing the centrifuge to reach 453 xg for two seconds before applying the brake. These brief pulse spins were repeated at least 4 times, or until examining the sample under a microscope revealed primarily epithelial organoid clusters and very few single cells. At this point, the organoid pellet was resuspended in 1 ml TRIzol and stored at -80°C until RNA isolation. High-quality total RNA (RIN > 7) was extracted from frozen mammary epithelial organoid pellets using NucleoSpin® miRNA isolation kit (MACHEREY-NAGEL) according to the manufacturer's protocol, isolating large and small (<200 nt) fractions separately. The “large” RNA fraction was used to prepare strand-specific poly-A+ RNA-seq libraries for sequencing. The “small” RNA fraction was used to make miRNA-seq libraries using NEXTflexTM small RNA-seq Kitv3.miRNA-seq library construction. Tissue samples (Additional file18) were collected similar to that described in previous section. QIAseq miRNA Library Kit (Qiagen, cat no. 331505) and QIAseq miRNA NGS 96 Index IL (Qiagen, cat no. 331565) kits were used to isolate miRNAs from all tissues except mammary gland. miRNAs from mammary gland were isolated using NEXTflexTM small RNA-seq Kitv3 (Illumina) according to the manufacturer's instructions. The isolated miRNA was subjected to 3' ligation to ligate a preadenylated DNA adaptor to the 3'ends of all miRNAs. An RNA adaptor was then ligated to the 5' end of the mature miRNA to complete 5' ligation. cDNA synthesis was completed using a reverse transcriptase (RT) primer containing integrated unique molecular identifiers (UMI). The RT primer bound to the 3' adaptor region and facilitated conversion of the 3'/5' ligated miRNAs into cDNA while a UMI was assigned to every miRNA molecule. After reverse transcription, a clean-up of the cDNA was performed using a streamlined magnetic bead-based method. Library amplification was accomplished by a universal forward primer from a plate being paired with 1 of 96 dried reverse primers in the same plate (Qiagen, cat no. 331565) to assign each sample a unique custom index. Following library amplification, a clean-up of the miRNA library was performed using a streamlined magnetic bead-based method. Libraries were then evaluated for quantity and quality measures before being normalized and pooled for Illumina sequencing.