GSM8080085: Sonicate fluid 69 [773]; Homo sapiens; RNA-Seq
Total RNA was extracted from sonicate fluid samples using the miRNeasy Serum/Plasma kit (QIAGEN, Germantown, MD) and analyzed for quantity and integrity using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Coon Rapids, MN) coupled with the Qubit RNA high-sensitive assay kit, and a 4200 TapeStation system (Agilent, Santa Clara, CA), respectively. RNA was treated with DNase (QIAGEN) and a Ribo-Zero rRNA removal kit (Bacteria) to remove genomic DNA and bacterial ribosomal RNA (rRNA). To prepare sequencing libraries, the Ovation SoLo RNA-Seq System (NuGEN Technologies) was used with 1 ng of RNA input. In addition, External RNA Control Consortium (ERCC) RNA Spike-In Mix 1 (Thermo Fisher Scientific)  was added as a control for library preparation and sequencing, according to the manufacturer's recommendations. The library construction process also included a human-specific rRNA depletion step that involved adding a custom-designed Solo AnyDeplete probe mix (NuGEN Technologies) after initial cDNA synthesis and amplification Sequencing was performed on an Illumina HiSeq 4000 instrument with 10 samples multiplexed per lane, with paired-end read and 100 cycle settings