Illumina NovaSeq 6000 sequencing; Investigating plasma circulating miRNA in dairy cows receiving essential fatty acids and/or conjugated linoleic acid during the transition to lactation
Investigating plasma circulating miRNA in dairy cows receiving essential fatty acids and/or conjugated linoleic acid during the transition to lactation
Blood sampling and plasma separation Blood samples were collected immediately after morning milking and before feeding, using the Vacuette system (Greiner Bio-One International GmbH, Kremsmünster, Austria) containing 1.8 g potassium-EDTA (K3EDTA)/L of blood. The plasma fraction was extracted after centrifugation at 1,500 × g (4 °C) for 20 min and stored at −80 °C. A detailed description of the animal, study design, feeding Management, diet composition, fatty acid composition of diets, and performance can be found in (Vogel et al., 2020, https://doi.org/10.3168/jds.2019-18065, and Gnott et al., 2020, https://doi.org/10.3168/jds.2020-18735). Cows were daily abomasally supplemented from d 63 antepartum (AP) until d 63 postpartum (PP) with coconut oil, providing no ALA or CLA (CTRL, n = 9; Bio-Kokosöl #665, Kräuterhaus Sanct Bernhard KG, Bad Ditzenbach, Germany); a combination of linseed oil (Derby Leinöl #4026921003087, Derby Spezialfutter GmbH, Münster, Germany) and safflower oil (Gefro Distelöl, Gefro Reformversand Frommlet KG, Memmingen, Germany) to provide an n-6/n-3 FA ratio of 1:3 in the supplement mixture (EFA, n = 9); Lutalin (CLA treatment, n = 10; cis-9,trans-11 and trans-10,cis-12 CLA, 10 g/d each; BASF SE, Ludwigshafen, Germany); or a combination of EFA and CLA (EFA+CLA, n = 10). The amounts of daily infused supplements are given in Table 1. Treatments were infused using 60-mL catheter-tip syringes twice a day (2 equal portions) at 0700 and 1630 h. All supplements were liquified by heating to 38°C to allow infusion. The fatty acid compositions of the added lipids are shown in Supplemental Table S1 (https://doi.org/10.3168/jds.2019-18065). During the dry period, each dose was halved. Sampling started at wk 10 AP and was terminated at wk 9 PP. RNA extraction and quality measurement Total RNA was extracted using the QIAGEN miRNeasy serum/plasma kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol (https://www.qiagen.com/de/resources/resourcedetail?id=46286f7e-0bee-4820-9af1-654b578c98c8&lang=en). The RNA yield and purity were assessed using an Agilent Small RNA kit on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Library preparation and next-generation sequencing  The QIAseq miRNA library kit (QIAGEN, Hilden, Germany) was used for library preparation following the manufacturer's instructions for serum/plasma samples (https://www.qiagen.com/jp/resources/download.aspx?id=d3efdc53-9f98-4632-9bfe-cb2efab7db79&lang=en). Final libraries were checked Caliper (PerkinElmer, Waltham, MA). Libraries were then prepared for next-generation sequencing (NGS) and sequenced on single-end 100 bp mode on the NovaSeq 6000 (Illumina, San Diego, CA) on the respective platform of IGA Technology Services, Udine, Italy (https://igatechnology.com/). Raw-sequencing data was checked for quality (format conversion to FASTQ, demultiplexing, Adapter Trimming, and UMI Remova) using the Illumina Bcl2fastq2 Conversion Software (version 2.20).