Fragmentation buffer

Total RNA of individual muscles was extracted using a standard Trizol Reagent Kit (Huayueyang Biotech Co. Ltd., Beijing, China), in accordance with the manufacturer’s protocol, and quantified using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). We then purified the mRNA from the total RNA (4 μg) using the RNA Purification Beads (Illumina, San Diego, CA, USA). The remaining RNA was cleaned three times using the Beads Binding Buffer (Illumina, San Diego, CA, USA) and the eluted RNA were incubated at 94 °C for 8 min. Fragmentation buffer (Illumina, San Diego, CA, USA) was applied to lyse the mRNA into fragments of a suitable size and the fragmented mRNA was used to construct a cDNA library using TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA). Afterward, A-Tailing Control (Illumina, San Diego, CA, USA) and Ligation Control (Illumina, San Diego, CA, USA) were applied to A-tailing and adapter ligation of the double stranded cDNA, respectively. Then, the cDNA libraries were diluted to 10 pM and quantified using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The libraries were sequenced on the Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) across one lane with paired-end 150 bp.

For each group, four biological replicates were selected, of which every two were combined into one. Then, the total RNA of tissue was extracted using TRIzol reagent (Invitrogen Corporation, CA, United States) in accordance with the manufacturer’s instructions. The Ribo-Zero rRNA Removal Kit (Illumina, Inc., CA, United States) was used to reduce the ribosomal RNA content of total RNAs. Then, the RNA was chemically fragmented into fragments of about 100 nucleotides in length using fragmentation buffer (Illumina, Inc.).

Poly-A+ RNA was selected from total RNA (1 μg) using oligo dT-attached magnetic beads. Bound RNA was fragmented by incubation at 94 °C for eight (8) minutes in 19.5 μl of fragmentation buffer (Illumina). First and second strand synthesis, adapter ligation and amplification of the library were performed using the Illumina TruSeq RNA Sample Preparation kit under conditions prescribed by the manufacturer (Illumina). Samples were tracked using “index tags” incorporated into the adapters. Library quality was evaluated using an Agilent DNA-1000 chip on an Agilent 2100 Bioanalyzer. Quantification of library DNA templates was performed using qPCR and a known-size reference standard. Cluster formation of the library DNA templates was performed using the TruSeq PE Cluster Kit v3 (Illumina) and the Illumina cBot workstation using conditions recommended by the manufacturer. Template input was adjusted to obtain a cluster density of 700–900 K/mm2. Paired end 50 base sequencing by synthesis was performed using TruSeq SBS kit v3 (Illumina) on an Illumina HiSeq 1000 using protocols defined by the manufacturer.

Total RNAs for five different stressors were equally pooled for the construction of cDNA libraries. Sequencing libraries were generated using the Illumina TruSeqTM RNA Sample Preparation Kit (Illumina Inc, CA, USA) following the manufacturer's recommendations. The purification of mRNA was performed using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in the Illumina proprietary fragmentation buffer. The cleaved RNA fragments were reverse transcribed into first strand cDNA using random oligonucleotides and SuperScript II. After second strand cDNA synthesis, fragments were end repaired, a-tailed, and indexed adapters were ligated. The products were purified and enriched by PCR to generate the final library. The library preparations were sequenced on an Illumina Hiseq 2000 platform to generate 100 bp paired-end reads.

Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, mRNA was purified using poly-T oligo attached magnetic beads. The mRNA was fragmented using divalent cations at high temperature in an Illumina proprietary fragmentation buffer. First-strand cDNA was synthesized using random oligonucleotides and SuperScript II. Second-strand cDNA was subsequently synthesized using DNA polymerase I and RNase H. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated, and the library fragments purified were using the AMPure XP system (Beckman Coulter, Brea, CA, USA). DNA fragments with ligated adaptors on both ends were selectively enriched using Illumina polymerase chain reaction (PCR) Primer Cocktail in a 15 cycle PCR reaction. The products were purified (AMPure XP system) and quantified using the Agilent high-sensitivity DNA assay with the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The sequencing libraries were sequenced using Illumina HiSeq.