GSM6312624: blood, Day 0, UGA4, 337; Homo sapiens; RNA-Seq
Total RNA was isolated from each blood sample in randomized batches using the PAXgene Blood miRNA Kit (QIAGEN, Hilden, Germany) according to the manufacturer's recommendations and stored at -80°C in a LoBind twin.tec 96-well PCR plate (Eppendorf, Hamburg, Germany). All samples were quantified with the Qubit BR RNA Assay on a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA) and their fragment size distribution was measured with an RNA Screentape on a 4200 TapeStation System (Agilent, Santa Clara, CA) Sequencing libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, Ipswich, MA) according to the manufacturer's protocols using either 1ug of input RNA or, in the case of several low-yield samples, the maximum possible input mass. AMPure XP Beads (Beckman Coulter, Brea, CA) were used in place of the supplied NEBNext Sample Purification Beads. Libraries were prepared in 3 batches with two negative library prep controls in each batch to monitor for reagent or sample-to-sample contamination. Each library was barcoded with NEBNext Multiplex Oligos for Illumina (NEB, Ipswich, MA) in a rotating scheme, which ensured that each pool had a unique set of barcodes. Each library was quantified with the Qubit dsDNA HS Assay on a Qubit 2.0 Fluorometer and fragment size was measured with an HSD1000 Screentape (Agilent, Santa Clara, CA) on a 4200 TapeStation System. All libraries made from experimental samples were of sufficient molarity for pooling and sequencing. A single sequencing library was prepared from a single biological sample. Libraries were then pooled with equimolar input at either 2nM or 3nM into sets of 96 libraries for RNA sequencing on a NovaSeq 6000 with the S1 2x150 - 300 Cycle configuration (Illumina, San Diego, CA) at the Genomics Core Facility (Center for Genomics and Systems Biology, New York University).