title: GSM6217227: PBMCs_3 without ALA and ConA; Canis lupus familiaris; RNA-Seq
library_construction_protocol: Total RNA was isolated from the PBMCs using the Maxwell RSC simplyRNA cells kit (Promega). Polyadenylated  mRNA was purified with oligo dT beads (NEBNext Poly (A) mRNA magnet Isolation Module, New England Biolabs, NEB). Complementary DNA (cDNA) libraries for Illumina sequencing were generated with NEBNext Ultra II RNA library Prep kit (NEB) and NEBNext Multiplex Oligos for Illumina (NEB). Extracted mRNA was incubated with NEBNext Random Primers in NEBNext First Strand Synthesis Reaction Buffer at 94°C for 15 min, and reverse transcription was performed with NEBNext Strand Synthesis Enzyme Mix. After reverse transcription, index sequences were inserted to cDNA fragments with PCR amplification using primers included in NEBNext Multiplex Oligos for Illumina. Following purification with AMPureXP beads (Beckman Coulter), the quality and the concentration of the libraries was evaluated using a Bioanalyzer. The confirmed libraries were mixed to equal molecular amounts against the clustering and sequencing on an Illumina NextSeq 500 DNA sequencer with 75 bp paired-end sequencing kit (Illumina).