Glucocorticoid Receptor

To conduct expression studies, neutrophils isolated from cows and calves were adjusted to 1x10⁶ live cells/ml. According to the manufacturer’s instructions, total RNA from blood neutrophils was extracted and purified using the TRIzol reagent (Invitrogen, Carlsbad, CA). RNase-Free DNase Set (Qiagen, India Pvt. Ltd.) was used to remove the genomic DNA contamination. The integrity of the RNA was evaluated by agarose gel electrophoresis (1.5% agarose), and the quantity and quality of RNA were verified by OD absorption ratio at λ₂₆₀/λ₂₈₀ using Bio Spec-nano (serial no., A116449; Biotech). A ratio of 1.9 to 2.0 was accepted as “pure” for RNA.
Gradient PCR was used to optimize the annealing temperature of each primer (including endogenous genes β-actin and GAPDH primer), followed by agarose gel electrophoresis (1.5%) to visualize the product size of PCR amplified products of target genes. Primers for specific bovine toll-like receptors (TLR2, TLR4), chemokine receptors (CXCR1, CXCR2), glucocorticoid receptor (GR-α), cluster of designation (CD62L, CD11b, CD25 and CD44) and endogenous genes (GAPDH, β-actin) were selected from the published literature (39 (link)–41 (link)) and shown in Table 2 (Sigma Chemicals Co., St. Louis, Missouri, USA). Quantitative real-time PCR (qPCR) (Roche’s Lightcycler 480) was carried out using Thermo Scientific Maxima SYBR Green qPCR Master Mix kit (Thermo Scientific, USA) in accordance with the manufacturer’s instructions. Briefly, template cDNA (1 μl), SYBR green (2x) mixes (5 μl), forward and reverse primers (0.5 μl each) and total reaction volume up to 10 μl was made using nuclease-free water. The protocol of RT-PCR consisted of 45 cycles at 95°C for 15 s, annealing at 58 or 59°C for 20 s, and performed denaturation kinetics to assess the reaction product. β-actin and GAPDH were used as endogenous genes and the mRNA abundance of the day -30 for cows and day of birth for calves was taken as a calibrator with which relative expression of all genes during different time points was estimated. The relative quantification of all genes was evaluated by the 2^−ΔΔCt method (42 (link)).

CRISPR/Cas9 KO of genes was performed as previously described^{96 (link)}. The guide RNAs targeting for human glucocorticoid receptor (GR) and androgen receptor (AR) are listed in Supplementary Table 1 and gRNAs and ZFNs for all glycoengineering performed were reported previously^{64 (link),97 (link),98 (link)}. Briefly, HEK293 cells grown in 6-well plates (60-80% confluency) were co-transfected with 1 µg of the respective gRNA and 1 µg GFP-tagged Cas9-PBKS using Lipofectamine 3000 (Invitrogen) following the manufacturer’s instructions. 24 h post-transfection, GFP-positive cells were bulk sorted and expanded for 7–8 days followed by single-cell sorting into 96-well plates using a cell sorter (SONY SH800). KO clones were selected by Indel Detection using Indel detection by amplicon analysis (IDAA)^{96 (link)} and final clones were further verified by Sanger sequencing, and indel sequences of used clones are listed in Supplementary Table 1.