Agilent bioanalyzer 2100 equipment

Total RNA was obtained from 100 mg of frozen intestinal tissues with the RiboPure kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol. RNA concentration and purity was calculated with a NanoDrop ND-1000 spectrophotometer (NanoDrop products, Wilmington, DE, USA). RNA integrity was checked with Agilent Bioanalyzer-2100 equipment (Agilent Technologies, Santa Clara, CA, USA) following the producer’s protocol. One microgram of total RNA was reverse-transcribed into cDNA in a final volume of 20 µL. The High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) and random hexamer primers were used, and the following thermal profile was applied: 25 °C, 10 min; 37 °C, 120 min; 85 °C, 5 s; 4 °C hold. A total of 25 ng of cDNA sample was pre-amplified using a 2× TaqMan PreAmp Master Mix and a 0.2× Pooled Taqman Gene Expression Custom Assays in a final volume of 10 µL. The thermal cycling conditions for the pre-amplification reactions were 10 min at 95 °C; 14 cycles of 15 s at 95 °C and 4 min at 60 °C; and a final step of 10 min at 99 °C. The pre-amplified cDNA product was stored at −20 °C until use.

Samples from the dorsal adipose tissue stored at −80 °C were used for the RNA extraction, following a modified version of the PureLink^TM RNA Mini Kit from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). The modification consists of an additional centrifugation of 10 min after homogenizing the sample with the polytron and taking the supernatant to another tube in order to improve the purity of the extract. RNA purity and quantification were checked with a Quawell NanoDrop Q9000 spectrophotometer (Quawell Technologies, Beijing, China). All samples were checked for integrity using Agilent Bioanalyzer-2100 equipment (Agilent Technologies, Inc., Santa Clara, CA, USA) and all of them passed the quality control test with an average higher than 7 RIN.

Total RNA was isolated from the LD muscle (100 mg) of 129 animals using the the RiboPure™ Isolation kit for High Quality Total RNA (Ambion^®; Austin, TX) following the manufacturer’s recommendations. RNA quantification and purity was done with a NanoDrop ND-1000 spectrophotometer (NanoDrop products, Wilmington, DE, USA). RNA integrity was checked by Agilent Bioanalyzer-2100 equipment (Agilent Technologies, Inc., Santa Clara, CA, USA), using only those samples with an RNA integrity number (RIN) greater than 7 for the RNA-Seq experiment.
Library preparation and sequencing was performed at CNAG institute (Centro Nacional de Análisis Genómico, Barcelona, Spain). For each sample, one paired-end library was prepared using TruSeq Stranded mRNA kit (Illumina, Inc.; San Diego CA, USA). To discriminate among samples, libraries were labeled by barcoding and pooled to be run in Illumina HiSeq 3000/4000 instruments (Illumina, Inc.; San Diego CA, USA). In brief, in this study 2 × 75 bp reads, a mean of 45.09 million of paired-reads per sample, and an average of 90.06% (ranging from 80.51 to 96.09%) of uniquely mapped reads were generated.