GSM3702048: P2T2-2-2; Homo sapiens; RNA-Seq
Total RNA extraction was carried out using the TRIZOL method The integrity of the total RNA samples for the preparation of the RNA-seq libraries was determined by Automated Capillary Electrophoresis using the Bioanalyzer 2100 system (Agilent Technologies, Inc., Santa Clara, CA) and the Agilent RNA 6000 Nano chip, the values of RIN obtained for the samples were >8. The mRNA was enriched by magnetic beads. The construction of the RNA-seq libraries was performed using the Illumina TruSeq Stranded mRNA sample prep kit (Illumina, Inc., SanDiego, CA). Subsequently, the RNA was fragmented in a range of  200-600 bp. Both ends of the double-stranded cDNA were repaired to subsequently bind the specific Illumin adapters, using T4 DNA polymerase and T4 DNA ligase, respectively. Finally, each library was amplified by PCR with specific oligonucleotides for the P5 and P7 adapters of Illumina and thus increase the number of molecules for the massive sequencing. Each library was analyzed by Automated Capillary Electrophoresis (Bioanalyzer 2100, Agilent Technologies, Inc., Santa Clara, CA) using the DNA High Sensitivity chip. Finally, the libraries were quantified using the Qubit® fluorometer with the Qubit ds DNA HS Assay kit (Invitrogen). The libraries were clonally amplified in the Flowcell and sequenced with the NextSeq 500 platform of the Illumina brand (Illumina, Inc., SanDiego, CA) and with the NextSeq 500/550 Mid Output kit v2 (150 cycles) with a run configuration Paired-End 2X76 indexed and following the protocol described by the manufacturer.