Preserved blood rna purification kit 1

Blood samples were collected in the quarantine nurseryat ~ 27 days of age, using Tempus Blood RNA Tubes (Thermo Fisher Scientific, USA) and then stored at − 80 °C until RNA extraction. The RNAs were isolated using Preserved Blood RNA Purification Kit I (Norgen, Canada) according to the manufacturer’s instructions. The RNA integrity number (RIN) of each extracted RNA was assessed by the 2100 Bioanalyzer (Agilent Technologies, USA) using the Eukaryote total RNA 6000 Nano kit. The RIN score was on average 7.9 and ranged from 4.1 to 9.9 (Table 2). WBC differentials were quantified on whole blood samples in K2 ethylenediaminetetraacetic acid (EDTA) tubes (Thermo Fisher Scientific, USA) taken at the same time, using the flow cytometry-based hematology analyzer (ADVIA®2120i Hematology System, Simens Healthineers, Germany) according to the manufacturer’s instructions [59 (link)]. The log2 transformed proportion of each WBC type was used to adjust gene expressions levels for blood cell composition (see later).

Total RNA was extracted from the Tempus tube samples from Replicate 2 of the treatment groups for both pig lines by using preserved blood RNA purification kit I (Norgen Biotek Corp, Thorold, Ontario, CA) per the manufacturer’s instructions. On-column DNA digestion was performed using DNase I (Qiagen, Valencia, CA, USA). Globin transcripts (HBB and HBA) were depleted by following an RNase H-mediated method [36 (link)]. The quantity and integrity of the RNA were monitored by using Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA) and Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) before and after globin depletion. The efficiency of globin depletion of each sample was checked by conventional RT-qPCR with ACTB and GAPDH as the internal controls. Globin depletion efficiencies for all RNA samples were above 85%. Metadata, including RNA integrity numbers (RINs) and concentration of RNA post globin depletion, CBC, and sequencing batches are available in Additional file 1: Table S1.

Peripheral blood for gene expression and DNA methylation analyses was sampled simultaneously in Tempus Blood RNA Tubes (Thermo Fisher Scientific, Waltham, MA, USA) and K2-EDTA tubes (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA), respectively. Total RNA was isolated from the Tempus Blood RNA Tubes using the Preserved Blood RNA Purification Kit I (Norgen Biotek, Thorold, ON, Canada) according to the manufacturer’s instructions. DNAse treatment was carried out as recommended. The quality and concentration of the RNA were measured using the BioAnalyzer 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA) and Qubit RNA HS (Thermo Fisher Scientific), with a mean RNA integrity number (RIN) of 9.2 and a concentration of 159 ng/μL. The total RNA samples were depleted for ribosomal RNA and globin transcripts with the Globin-Zero^® Gold rRNA Removal Kit (Illumina, San Diego, CA, USA).
DNA was isolated from the K2-EDTA tubes using the Maxwell 16 Cell LEV DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The quality and concentration of the DNA were measured using Nanodrop (Thermo Fisher Scientific). Bisulfite conversion of DNA (500 ng) was done using the EZ-96 DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions.