title: Illumina HiSeq 2500 paired end sequencing; RNAseq of circulating neutrophils in peripartal grazing dairy cows divergent in metabolic health
design: RNAseq of circulating neutrophils in peripartal grazing dairy cows divergent in metabolic health
library_construction_protocol: Blood was sampled from the coccygeal blood vessels for neutrophil isolation at three time points over the transition period (day 0, week 1, and week 4 post-calving). Dairy cows were grazed outdoors at Scott Farm, Hamilton, New Zealand (37°46’S 175°18’E) between July and October 2013. Cows were offered a diet of fresh pasture (average dry matter intake [DMI] = 12.4 ± 0.8 kg/d), supplemented with corn silage (average DMI = 0.8 ± 0.8 kg DM/d), and pasture silage (average DMI = 1.1 ± 1.0 kg DM/d) post-calving.  Total RNA extracted using Trizol method (Life Technologies, Auckland, New Zealand), DNase treated (Ambion), and checked for quality using capillary gel electrophoresis electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies, CA, USA). Quantity was analysed using the Qubit ® Fluorometer and the RNA broad range assay kit (Life Technologies, New Zealand). Library preparation for Illumina HiSeq (2 x 125 bp PE reads) using V4 chemistry. Total RNA TruSeq stranded libraries included Ribo-Zero ribodepletion and ERCC spike-in controls. Average library size (± SD) was 339 ± 17 bp, with the exception of Sample21. Libraries were sequenced on an Illumina HiSeq 2500 (Illumina). Library construction and sequencing was completed by New Zealand Genomics Ltd., Dunedin, New Zealand.
attributes.tag: Experimental Factor: phenotype
attributes.value: high risk
attributes.tag: Experimental Factor: time
attributes.value: 0