Pltlts_Long_161_187_TtlRNA_18Apr2012
The study was approved by the Institutional Review Board of Thomas Jefferson University and informed consent was obtained for all participants. Peripheral blood samples were collected from 10 healthy males with no previous history of thrombosis or history of bleeding. The median age of the subjects was 24.5 years. Five of the donors were White and five were Black (Supp. Table 1). RNA was extracted from highly purified leukocyte- depleted platelets (LDPs) using magnetic beads (Miltenyi Biotec) against CD45 for leukocyte depletion and CD235 to select red blood cells. This protocol yielded a purity of less than 1 leu- kocyte to 1 million platelets. RNA extraction from LDPs was performed with TRIzol (Invitrogen). In Bray et al13, we showed that ribosomal RNA (rRNA) depletion from platelet RNA preparations impacts adversely and non-uniformly on the relative abundance of transcripts. Consequently, we sequenced total RNA, as an rRNA depletion step would have skewed the estimates of rela- tive abundance of the various molecular categories.    Total RNA sequence library construction, emulsion PCR, and sequencing runs were performed following the Applied Biosystems/Life Technologies protocols, and se- quencing was performed on the SOLiD 5500xl platform. The total RNA was size selected and for each sample, long RNA libraries between 150 and 500 nucleotides (nts) were generated and 50 nt reads were sequenced using a single-end approach. No multiplexing was used.
Thomas Jefferson University
Pltlts_Long_161_187_TtlRNA_18Apr2012