Rneasy plus mini extraction kit

RNA was extracted using the RNeasy plus mini extraction kit (Qiagen) and QIAshredders (Qiagen). The tube containing the swab in RNAlater was thawed and placed in 600ul of extraction buffer using sterile forceps. The swab and buffer were vortexed at maximum speed for 2 minutes before being placed into a QIAshredder and centrifuged at maximum speed for 5 minutes. Avoiding the cell debris pellet the flowthrough was retained and used in the extraction following the standard protocol in the kit. The RNA was eluted into 50ul of sterile water.
To increase the concentration of RNA, extractions were randomly pooled into groups of 3–5 animals for sequencing, using 25ul of each extraction in each pool. This pooled RNA was concentrated using the NucleoSpin RNA Clean-up XS, Micro kit for RNA clean up and concentration (Machery-Nagel). The concentrated RNA was eluted into 20ul of sterile water. The Stranded Total RNA Prep with Ribo-Zero Plus (Illumina) kit was used to prepare the cDNA libraries for paired-end sequencing on the Illumina Novaseq 6000 platform. Using two lanes, each sample was sequenced once on each lane and the reads were combined from both lanes for each sample.

RNA was extracted from thawed cryopreserved PBMC samples using the RNEasy plus Mini extraction kit (Qiagen) per manufacturer’s instructions or from PAXgene samples using the SimplyAmp extraction kit (Promega) using the Tecan Freedom EVO 150 automated system. Expression of mRNA transcripts comprising the 11-gene ACS COR signature (Darboe et al., 2018 (link)) was measured using commercial TaqMan primer/probe sets (Thermo Fisher Scientific; Supplementary Appendix A1) by microfluidic qRT-PCR using 96.96 Gene Expression chips (Fluidigm) on the Biomark HD multiplex instrument (Fluidigm) after cDNA synthesis using Superscript II reverse transcriptase (Invitrogen), as previously described (Zak et al., 2016 (link)). Signature score data for the 11-gene ACS COR signature from the CTBC, TRUTH and IMPRESS cohorts are available in Supplementary Tables S1–S3. Signature scores for the 11-gene ACS COR signature were calculated using in-house scripts written in R as previously described (Zak et al., 2016 (link); Darboe et al., 2018 (link)), and provided as an Excel template (Supplementary Tables S4, S5).

RNA was extracted from whole ovaries or tumour pieces using RNeasy Plus mini extraction kit (Qiagen, Germany) and reverse transcription performed using QuantiTect Reverse transcription kit (Qiagen, Germany). All quantitative real time rtPCR assays were carried out three times using TaqMan® universal master mix II with UNG (Applied Biosystems, USA), Taqman® assays (Additional file 1: Table S3) and QuantStudio™ 7 Flex Real Time PCR system (ThermoFisher, USA), and relative expression levels determined using QuantStudio™ 7 Real Time PCR software.