GSM8818659: REDUC_34 PBMC; Homo sapiens; RNA-Seq
Total RNA was extracted from PBMCs (CLEAR, TEACH and REDUC) or CD4+ T cells (ACTG A5345) using the Qiagen RNeasy Micro Kit per manufacturer's protocol. RNA yields ranged from 300 ng to 3,500 ng. RNA quality was tested using a Qubit 2 fluorometer (Thermo Scientific). Sequencing libraries were prepared by Azenta Life Sciences employing the NEB Next Ultra II RNA Library Prep Kit for Illumina.  Briefly, mRNA was enriched using oligo (dT) beads, then fragmented for 15 minutes at 94°C followed by cDNA synthesis. Fragments were then end-repaired and adenylated. Universal adapters were ligated to cDNA fragments, then indexes were added with library enrichment by PCR. RNA integrity and library size was assessed by Agilant Tapestation, and concentrations were determined by Qubit 2 fluorometer with final quantification by qPCR. Flow cells were prepared with multiplexed and clustered sequencing libraries, then loaded onto an Illumina HiSeq instrument.