GSM5213700: BC-184_S15; Homo sapiens; RNA-Seq
Peripheral blood (30 ml) was drawn from the study subjects by venipuncture. Plasma was collected by centrifugation, and subsequently leukocytes and erythrocytes were separated using LeucosepTM tubes according to the manufacturer's instructions (Greiner Bio-One Inc., Monroe, NC). The collected plasma, and leukocyte and erythrocyte fractions were aliquoted into multiple microtubes (Eppendorf, Inc., San Diego, CA), snap frozen, and preserved at -80° C until further analysis. An aliquot of the thawed leukocyte samples from each subject was used for total RNA isolation using the RNeasy Mini Kit (Qiagen, Valencia, CA). Libraries for RNA-seq were prepared from total RNA (300 ng per sample) using the KAPA Stranded mRNA-Seq Kit (Kapa Biosystems, Inc. acquired by Roche). The workflow consisted of mRNA enrichment and fragmentation, first strand cDNA synthesis using random priming, followed by second strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (ds cDNA), and incorporation of dUTP into the second cDNA strand. cDNA generation was followed by end repair to generate blunt ends, A-tailing, adaptor ligation, and PCR amplification. Different adaptors were used for multiplexing samples in one lane.