Ribo zero globin

Serum was collected from 23 eligible patients at TP0, TP1 and TP2 for responders, or at the time the patient went off treatment if before cycle 6.
RNA sequencing: The buffycoat samples were collected from ovarian cancer patients at TP0, TP1, and TP2. RNA was isolated from the buffy coat samples using Direct-zol RNA MiniPrep Plus (Zymo Research, Irvine, CA). Total RNA was tested for suitable mass (PicoGreen RNA) and integrity (Agilent TapeStation), converted to cDNA (Illumina TruSeq), depleted of ribosomal and globin RNA (Illumina RiboZero Globin), and sequenced on an Illumina HiSeq 4000 instrument in the UCLA Neuroscience Genomics Core Laboratory, following the manufacturers’ standard protocols. All serum samples were processed in duplicate and biomarker analysis was performed by those blinded to the outcome.

Total RNA was isolated from patient blood using the RNeasy Mini Kit (Qiagen; catalog no. 74104) according to the manufacturer’s protocol.RNA was digested using RNase-free DNase I to remove any contaminating genomic DNA and quality was assessed by the RIN using the Agilent 2100 Bioanalyzer. Library preparation was performed using Illumina TruSeq Stranded Total RNA with Ribo-Zero Globin/Illumina TruSeq Stranded mRNA (using poly-A selection). Approximately 100 million reads were generated per sample on the Illumina Novaseq. The sequencing reads were demultiplexed and the fastq files were aligned to the hg19 reference genome (NC_000001.10) using the STAR aligner (v. 2.5.3a) (Dobin et al., 2013 (link)). The duplicate marking was performed using Picard Tools (v. 2.18.4), RNA-SeQC v1.1.8. was used for quantifying postalignment quality metrics.