Gaiix flow cell

We used Illumina’s Genome Analyzer IIx (GAIIx) for mRNA-sequencing by loading one sample per lane. For mRNA-sequencing, the library was diluted to 10 nM in EB buffer and then denatured using the Illumina protocol. The denatured libraries were diluted to 12 pM, followed by cluster generation on a single-end Genome Analyzer IIx (GAIIx) flow cell (v4) using an Illumina cBOT, according to the manufacturer's instructions. The Illumina GAIIx flow cell was run for 75 cycles using a single-read recipe (v4 sequencing kits) according to the manufacturer’s instructions.

Polyadenylated RNA samples from adult male and female S. japonicum parasites were isolated from total RNA using oligo-(dT) conjugated magnetic beads (Dynabeads®, Invitrogen, CA, USA). The mRNA was interrupted into short fragments by adding the fragmentation buffer provided by the manufacturer (Illumina RNA-seq kit, part no. 1004898). With these short fragments as templates, random hexamer primers were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I, respectively. Short fragments were purified following instructions accompanying the kit (QiaQuick PCR Purification Kit, Qiagen, Germany), and double-stranded cDNAs were end-repaired according to manufacturer-recommended protocols, followed by connection with Illumina adapters (Illumina RNA-seq kit, part no. 1004898). The fragments were first amplified by PCR. Purified cDNA fragments were pooled and indexed and loaded onto one lane of an Illumina GA IIX flow cell. A total of 75 pair-end sequencing cycles were carried out. Cluster formation, primer hybridization, and pair-end sequencing were performed according to the provided protocols
[25 (link)].

To obtain RNA-seq gene expression data, we used the Illumina Genome Analyzer IIx (GAIIx) platform (San Diego, CA, USA) to sequence the poly-adenylated fraction (mRNA) of 11 tumor samples (4 SBTs, 3 stage II HGSOCs, and 4 stage III HGSOCs). Using a Covaris, Inc. E210 ultrasonicator (Woburn, MA, USA), we fragmented sample mRNA before using it as a template for first-strand cDNA synthesis using random primers and Life Technologies, Inc. SuperScript II Reverse-Transcriptase (Carlsbad, CA, USA). We ligated Illumina adaptors to the ends of double-stranded cDNA fragments and then used the Sage Science Pippin Prep system (Beverly, MA) to select 500-bp final products. After size selection, library enrichment consisted of 10 to 12 PCR cycles. Using version 5 chemistry, we sequenced each library on a single lane of an Illumina GAII_x flow cell and used the Illumina pipeline for image analysis and base-calling. These methods resulted in 40 to 69 million 51-bp paired-end reads (median: 65 million reads) per tissue sample.