Truseq pe cluster kit v3 cbot hs kit

Total RNA was extracted from 100 mg of frozen LD muscle that was collected at slaughter using the TRIzol reagent (Life Technologies, Carlsbad, CA). RNA integrity was verified by Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Only samples with RIN > 8 were used. A total of 2μg of total RNA from each sample was used for library preparation according to the protocol described in the TruSeq RNA Sample Preparation kit v2 guide (Illumina, San Diego, CA). Libraries average size was estimated using the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantified using quantitative PCR with the KAPA Library Quantification kit (KAPA Biosystems, Foster City, CA, USA). Quantified, samples were diluted and pooled (three pools of six samples each). Three lanes of a sequencing flowcell, using the TruSeq PE Cluster kit v3-cBot-HS kit (Illumina, San Diego, CA, USA), were clusterized and sequenced using HiScanSQ equipment (Illumina, San Diego, CA, USA) with a TruSeq SBS Kit v3-HS (200 cycles), according to manufacturer instructions. The sequencing analyses were performed at the Genomics Center at ESALQ, Piracicaba, São Paulo, Brazil.

Total RNA of samples from the collected tissue section was extracted immediately after collection using TRIzol Reagent (Life Technologies, Carlsbad, CA) extraction protocol. The integrity of RNA samples was evaluated with a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), and only samples with RIN (RNA Integrity Number) values > 7 were used for subsequent analyses. RNA libraries were prepared with 2 µg of total RNA using the TruSeq RNA Sample Preparation kit v2 (Illumina, San Diego, CA) according to the protocol provided by the manufacturers. Average library sizes were estimated using an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and quantification was performed by quantitative PCR with the KAPA Library Quantification kit (KAPA Biosystems, Foster City, CA, USA). One lane of a sequencing flow cell, using the TruSeq PE Cluster kit v3-cBot-HS kit (Illumina, San Diego, CA, USA), was clustered and sequenced with TruSeq SBS Kit v3-HS (200 cycles) equipment, according to the manufacturer’s instructions. Sequencing was performed using HiSeq 2500 (Illumina, San Diego, CA, USA), using a paired-end (2 × 100 bp) protocol. All sequencing steps were performed at the ESALQ-USP Animal Genomics Center, located in the Animal Biotechnology Laboratory of ESALQ-USP, Piracicaba, São Paulo, Brazil.

Total RNA was extracted for each sample with TRIzol® reagent (Life Technologies, Carlsbad, CA, USA) from 100 mg of frozen LT muscle. RNA integrity was verified by Agilent 2100 Bioanalyzer® (Agilent, Santa Clara, CA, USA), where only samples with RIN > 8 were used. A total of 2 μg of RNA from each sample was used for library preparation according to the protocol described in TruSeq RNA Sample Preparation kit® v2 guide (Illumina, San Diego, CA, USA). The resultant libraries were quantified using a KAPA Library Quantification kit® (KAPA Biosystems, Foster City, CA, USA), according to Illumina's library quantification protocol. Finally, libraries were pooled (six pools of eight samples each) to perform multiplexing sequencing process, which adds an individual barcode sequences to each sample allowing that each one can be distinguished and analyzed separately during the data analysis. Six lanes of a sequencing flowcell, using the TruSeq PE Cluster kit v3-cBot-HS kit (Illumina, San Diego, CA, USA), were clustering and sequenced using HiSeq (Illumina, San Diego, CA, USA) with a TruSeq SBS v3-HS Kit (200 cycles), according to manufacturer’s instructions. Paired-end reads of 2 × 100 bp were produced. The sequencing analyses were performed at the Genome Center at ESALQ, Piracicaba, São Paulo, Brazil.