Agilent 2100 bioanalyzer device

Cell pellets were suspended in TE buffer (10 mM Tris, pH 8.0; 1 mM EDTA) containing 1 mg/mL lysozyme (Sigma, Darmstadt, Germany) and total RNA was isolated from lysed cells using TRI reagent (Sigma) according to manufacturer’s protocol. Removal of DNA was achieved by treatment of samples with TURBO DNA-free kit (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality and quantity were determined by agarose gel electrophoresis and using the Nanodrop 2000 machine (Thermo). Furthermore, the RNA quality was assessed at the sequencing facility of the Institute of Advanced Biotechnology (AIB) on an Agilent 2100 Bioanalyzer device. All samples displayed RNA integrity numbers higher than 9.

The Agilent 2100 Bioanalyzer device (Agilent Technologies, Santa Clara, CA, USA) was used to assess the integrity of the RNA. The RNA integrity value (RIN) of the samples ranged between 7.1 and 9. Paired-end libraries with fragments of 300 bp were prepared using the True-Seq RNA-Seq sample preparation Kit v2 (Illumina, San Diego, CA, USA). The fragments were sequenced on an Illumina Hi-Seq 2000 sequencer (Fasteris SA, Plan-les-Ouates, Switzerland), according to the manufacturer’s instructions at CNAG (Centro Nacional de Análisis Genómico, Barcelona, Spain), generating paired-end reads of 75 bp.

For RNA preservation, the sampled tissues were preserved in an RNA-stabilization solution (Ambion RNAlater; Life Technologies) and stored at 4°C for 24 h. Subsequently, the RNA-stabilization solution was removed, and the samples were frozen at -80°C until RNA extraction. RNA was extracted using the miRNeasy Mini KIT (Qiagen, Germany) with adaptations for use in adipose tissue (up to 100 mg of tissue and inclusion of Qiagen RNeasy Lipid Tissue Mini Kit). The Agilent 2,100 Bioanalyzer device (Agilent Technologies, CA, USA) was used to estimate the RNA integrity value, which was higher than 7 for all the samples. The UltraTM RNA Library Prep Kit (NEBNext^®, MA, USA) was used for cDNA library construction at Novogene in Cambridge (UK). The 12 cDNA libraries were sequenced on an Illumina Novaseq 6,000. A minimum depth of 30 million 150 bp stranded paired-end reads was generated for each sample. The raw datasets derived from the sequencing are available at ArrayExpress repository with reference E-MTAB-12130.

Samples of LD of the selected 12 pigs were collected at slaughter, frozen in liquid nitrogen and stored at -80°C until analyzed. Total RNA was extracted using RiboPure^TM of High Quality total RNA kit (Ambion, Austin, TX, United States) following the manufacturer’s recommendations. The RNA integrity was assessed using the RNA Integrity Number (RIN) value from the Agilent 2100 Bioanalyzer device (Agilent technologies, Santa Clara, CA, United States). RNA integrity values ranged between 7 and 8.

Muscle samples were maintained at −80 °C until gene expression analysis. For the transcriptome study, 36 muscle samples were used, corresponding to 12 Iberian animals belonging to the first slaughter (6 animals of each diet group) and 24 animals belonging to the second slaughter (12 animals of each breed, with 6 animals corresponding to each dietary group). The animals were randomly selected to perform transcriptomic analysis, representing all available litters. Total RNA was isolated from 50–100 mg samples of BF using the RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer’s recommendations. The obtained RNA was quantified using NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was assessed with an Agilent 2100 bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA) and submitted to the CNAG_CRG (Centro Nacional de Análisis Genómico, Barcelona, Spain). Libraries were prepared using the TruSeq mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801, San Diego, CA, USA) according to manufacturer’s protocol. Each library was paired-end sequenced (2 × 75 bp) using TruSeq SBS Kit v3-HS, on a HiSeq2000 platform (Illumina, San Diego, CA, USA).

Total RNA was isolated using TRI Reagent (Sigma, Darmstadt, Germany) according to manufacturer’s protocol. Removal of DNA was achieved by treatment of samples with TURBO DNA-free kit (Thermo Fisher Scientific). RNA quality and quantity was determined by agarose gel electrophoresis and using the Nanodrop 2000 machine (Thermo, Carlsbad, CA, USA). Furthermore, the RNA quality was assessed at sequencing facility (Vienna Biocenter Core Facility, NGS unit) on an Agilent 2100 Bioanalyzer device. All samples displayed RNA integrity numbers higher than 9.

For the transcriptomic study, 24 animals were used (6 animals of each breed, corresponding to each diet group). Total RNA was isolated from 50–100-mg samples of subcutaneous ham fat using the RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer’s recommendations. The obtained RNA was quantified using NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was assessed with an Agilent 2100 bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA) and submitted to the Centro Nacional de Análisis Genómico (CNAG-CRG; Barcelona, Spain). Libraries were prepared using the TruSeq mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801, San Diego, CA, USA) according to the manufacturer’s protocol. Each library was paired-end sequenced (2 × 75bp) by using TruSeq SBS Kit v3-HS in a HiSeq2000 platform (Illumina, Inc.).