library_construction_protocol: RNA was extracted from frozen PBMC pellets using TRIzol according to the manufacturer's recommendations. RNA pellets were resuspended in 20 uL of 1:40 RNAse inhibitor/RNAse-free water mix and stored at -80°C. A NanoDrop C Spectrophotometer was used for RNA quantification measuring absorbance at 260 nm. Samples were assessed for quality based on AD 260/280 ratios of > 1.9 and OD 260/230 > 2.0.  Finally, quality of RNA samples was confirmed using a Tapestation 4200 (Agilent, Inc). RNA samples having a RIN > 7 were used for subsequent analysis. A targeted RNA sequencing approach was used to quantify expression levels in PBMC from the coccygeal vein of previously diagnosed PI calves vs. controls. Primers were selected to produce amplicons that span exon-intron boundaries, using gene models extracted from Ensembl (www.ensembl.org) based on the cow genome assembly ARS-UCD1.2. The primer sets information is provided in Additional Table 2. After validating the primers set, cDNAs were amplified in triplicates and sequenced using the same multiplex approach. Targeted RNA-seq was performed by an optimized Hi-Plex approach by Floodlight Genomics (Knoxville, TN, USA) as part oftheir Educational and Research Outreach Program (EROP) and sequencing on an Illumina MiSeq device running a 2 × 150 configuration. The resulting sequences were mapped to the Bos Taurus genome assembly using CLC Genomics Workbench version 9.5.2 (Qiagen; Bethesda, MD) with default settings.  Multi-dimensional scaling (MDS) showed a separation in PI status compared to the control animals with the remove unwanted variation factor (RUV) set to 4. Targeted RNAseq