Blood samples and RNA isolation
Animal protocols were approved by the Kansas State University and University of Alberta Animal Care and Use Committees. A total of 24 blood samples were used to conduct two independent studies: comparisons of three GR methods to select the best method and evaluating the effects of the selected GR method on an RNA-seq experiment. For the first study, 3 mL of blood samples from 9 pigs of 1-2 months age produced from Landrace x Large White selected from a commercial populations used in the Porcine reproductive and respiratory syndrome Host Genetics Consortium (PHGC) studies  were collected in TempusTM Blood RNA tubes (Life Technologies, Carlsbad, CA, USA) and 2.5 mL of blood samples from 3 crossbred pigs of Large White x Landrace were collected in PAXgeneTM Blood RNA tubes (PreAnalytiX, Qiagen, Germany) at the University of Alberta. Total RNA was isolated with PAXgeneTM Blood RNA Kit (PreAnalytiX, Qiagen) for PAXgeneTM tubes and either TempusTM Spin RNA Isolation Kit (Life Technologies, Carlsbad, CA, USA) or magnetic bead based MagMaxTM for Stabilized Blood Tubes RNA Isolation Kit for Tempus tubes (Life Technologies), according to the respective manufacturer’s instructions.
For the second study evaluating the effects of RNase H mediated GR protocol on RNA-seq, another set of 12 blood samples were drawn from crossbred pigs of Duroc x (Landrace x Yorkshire) in a PHGC population. Three mL of blood from each pig at 1-2 months of age was collected into TempusTM Blood RNA Tubes at Kansas State University. Total RNA was isolated using the MagMaxTM for Stabilized Blood Tubes RNA isolation kit according to the manufacturer’s protocol.
RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Nano-Drop Technologies, Wilmington, DE, USA) and RNA quality was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). To determine an accurate 28S/18S rRNA ratio in the pig, we aligned the human 28S sequence against pig genome build 10.2 using BLAST and identified 97-100% of similarity on pig chromosome 6: 871128-866484 (Ensembl release 73). The sizes of the 28S and 18S genes in pig were estimated to be 4645 bp and 2302 bp, respectively, yielding an rRNA ratio of 2.02, whereas the rRNA ratios in human and mouse are known to be 2.69 and 2.53, respectively (the ratio obtained from Genbank database; M11167 and X03205 in human and NR003279 and NR003278 in mouse).
Design of porcine specific oligonucleotides
We first tested GLOBINclearTM Human Kit (Ambion, Austin, TX, USA) which hybridized biotinylated oligonucleotides with globin transcripts by binding to Streptavidin Magnetic Beads. Second, we designed porcine specific biotinylated Peptide Nucleic Acids (PNA) oligos to inhibit reverse transcription of globin transcripts (HBA: 5′-CGAGGCTCCAGCTTA-3′ and HBB: 5′-CACCAGCCACCACCT-3′). Third, we designed four porcine specific antisense oligonucleotides for HBA and HBB using Primer 3 (v. 0.4.0) (Table 1) to hybridize with globin transcripts prior to digestion with RNase H. To design porcine specific oligonucleotides, we first used Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) to align the porcine HBA (ENSSSCT00000008741) and HBB (ENSSSCT00000016076) transcript sequences in the current assembly of the pig genome (build 10.2) with their orthologues from human, mouse, cow and pig obtained from the Ensembl database (http://www.ensembl.org) and then checked the similarity of the 3′ end hybridization sites (Additional file 1: Figure S2).
Globin reduction treatment
GR treatment with porcine specific oligonucleotides was performed using a modified Affymetrix GR protocol . In brief, 10X GR oligonucleotides mix was prepared adding 100 uL each of two HBA Oligos at 30 uM, two HBB Oligos at 120 uM per reaction, yielding a final concentration of 7.5 uM HBA Oligos and 30 uM HBB Oligos. Three ug of denatured total RNA was hybridized in a thermal cycler at 70°C for 2 min with the 400 uL 10X GR oligonucleotides mix in hybridization buffer (100 mM Tris-HCL, pH 7.6; 200 mM KCl) at 70°C for 5 min, then cooled to 4°C. The RNA-DNA hybrids were digested with 2 U RNase H (Ambion) in the reaction buffer (100 mM Tris–HCl, pH 7.6, 20 mM MgCl2, 0.1 mM DTT, SUPERase-In) at 37°C for 10 min and cooled to 4°C. The reaction was stopped by addition of 1.0 ul 0.5 M EDTA. RNase H treated RNA was immediately purified with the RNeasy MinElute Cleanup Kit (Qiagen, Toronto, Canada, Cat. No.: 74204) according to manufacturer’s instructions. RNA quality of GR treated samples was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.).
Quantitative real-time PCR (qPCR) analysis
We quantified the mRNA level of the porcine HBA and HBB transcripts by SYBR Green I based qPCR using a StepOneTM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). First strand cDNA was synthesized using SuperScript® II reverse transcriptase (Invitrogen) and random hexamer primers in a final volume of 20 μL following the manufacturer’s instruction. SYBR Green I based qPCR was performed in a total volume of 10 μL per reaction comprising 2 μL of template, 1 μL of the assay-specific primer mix, 5 μL of the Fast SYBR® Green Master Mix Bulk Pack (Applied Biosystems) and 2 μL of water. The reaction conditions used were one cycle of 95°C for 3 min for initial denaturation, 23 cycles of 95°C for 30 s and 60°C for 30 s. The primer sequences are shown in Additional file 1: Table S1.
Library preparation for sequencing
Poly-A + fractions from the GR treated samples and respective non GR treated samples (1.5 μg RNA each) were purified by using oligo-dT magnetic beads (Illumina, Inc., San Diego, USA), and used to construct cDNA libraries. The Poly (A+) RNA was primed with random hexamers and fragmented at 94°C for 8 min. Second strand cDNA was synthesized after the first strand cDNA using SuperScript II (Invitrogen). The cDNA fragments were end-repaired and a single ‘A’ nucleotide was added to 3′-ends to prevent them from cross ligation during the adapter ligation step. Then individual RNA adapter index oligos were ligated to the end-repaired cDNA and subsequently amplified using Veriti Thermo cycler (Applied Biosystems). The initial denaturation was performed at 98°C for 30 seconds, followed by 15 cycles at 98°C for 10 seconds, annealing at 60°C for 30 seconds and extension at 72°C for 30 seconds. The final extension was followed at 72°C for 5 minutes, and held at 10°C.
The quality and size (~260 bp) of the resulting cDNA libraries were assessed using the High sensitivity DNA Kit (Agilent Technologies, Inc.) in an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). The quantification was performed using StepOneTM Real-Time PCR System (Applied Biosystems), as suggested in the Sequencing Library qRT-PCR Quantification Guide (Illumina, Inc.). The KAPA SYBR® FAST ABI Prism qPCR Kit (Kapa Biosystems, Inc., Woburn, USA) was used for the qPCR reactions. The individual libraries were pooled into 2 nM after quantification.
Sequencing was performed on the HiSeq System (Illumina, Inc.). The pooled 10 μL of the 2 nM libraries were diluted and denatured. The pooled cDNA libraries (12 pM) were loaded on the cBot (Illumina, Inc.) for clustering on a flow cell, and single-read cluster generation proceeded using the TruSeqTM SR Cluster Generation Kit v3 (Illumina, Inc., Cat.: FC-930-3001). A portion of each library was diluted to 10 nM and stored at -20°C. Fifty cycles of sequencing-by-synthesis using the paired-end protocol was performed on a HiSeq (Illumina, Inc.) according to manufacturer’s instructions. Real-time analysis and base calling was performed using the HiSeq Control Software Version 1.4.8 (Illumina, Inc.).
Sequence reads with base quality scores were produced by the Illumina sequencer. Raw reads were processed using the Illumina CASAVA (v. 1.8) to filter out the low-quality reads. Sequence reads were then aligned to the pig genome reference assembly (build 10.2; ) using TopHat 2.0.8  with default parameters. The number of reads uniquely mapped to each gene (Ensembl 71 annotation) was determined using Htseq-count (v0.5.3.p3; ). To determine number of genes identified in each sample, we required a read count >5.
To identify genes detected at decreased or increased levels between the globin reduced and non-reduced samples, the read count data were analysed using edgeR (version 3.0.8)  in R (version 2.15.2), as described . Count data was normalized by the library size to account for different numbers of reads obtained from each sample. To determine differences in detection levels between the two groups, an exact test for the negative binomial distribution was used. The genes were considered to be differentially detected at FDR <0.05. RSeQC (v2.3.3)  was used for read distribution over gene body to check 5′/3′ bias. We used BlastN (v2.2.25)  to perform the alignment between globin oligos and the genes with decreased levels after GR treatment.