Ercc rna spike in control mix 1

RNA-seq libraries were prepared for each time-point and steady-state sample using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) automated on the NGS WorkStation (Agilent) at The Centre for Applied Genomics (TCAG) according to the manufacturer’s instructions. PCR cycle numbers were determined using the PCR Add-on Kit for Illumina (Lexogen). All steady-state samples were processed with 250 ng of total RNA input. To minimize variability between time-points within a batch, RNA samples were processed with the same total RNA input with a minimum of 100 ng of total RNA used. Each sample was spiked-in with ERCC RNA Spike-In Control Mix 1 (Ambion) according to the manufacturer’s instructions prior to the start of library preparation. Library quality and quantity were measured at TCAG prior to sequencing with Bioanalyzer (Agilent) and KAPA qPCR (Roche). Sequencing was also performed at TCAG on the Illumina HiSeq 2500 with single-end 100 bp read length yielding 40 to 50 million reads. Datasets can be accessed from GEO using the access number GSE191168.

ERCC RNA Spike-in Control Mix 1 was purchased from Life Technologies (Carlsbad, CA). It was further diluted 1:100 in the background of human normal universal RNA (BioChain, CA). 10 ng total RNA containing the ERCC RNA were reverse transcribed into cDNA using QuantiTect Reverse Transcription kit (QIAGEN, Germany). One fifth of the cDNA was used in the barcode assignment step together with 2nM each BC primer, 16 mM Mg²⁺, 6U HotStarTaq and 1X miScript preamp buffer. The following barcode assignment conditions were used: 95 °C for 15 min, 55 °C for 15 min, 65 °C for 15 min, and 72 °C for 7 min. To ensure complete removal of excess BC primers, reaction was purified in two rounds using GeneRead Size Selection Kit. The purified DNA was then mixed in 25ul with 2nM each non-BC primer, 4 mM Mg²⁺, 0.45 mM dNTP, 6U HotStarTaq and 1X miScript preamp buffer. The reaction was continued at following conditions: 95 °C for 15 min; 20 cycles of 95 °C for 15 s and 55 °C for 5 min; 98 °C for 15 min. After that, universal adapter primers, new HotStarTaq and buffers were added in proportion to bring the reaction volume to 50ul. The reaction was further incubated at the following conditions: 95 °C for 15 min; 26 cycles of 95 °C for 15 seconds and 60C for 2 min. Resulting DNA libraries were purified using GeneRead Size Selection Kit, and quantified using GeneRead DNAseq Quantification Kit.

Total RNA was extracted from DTX-sensitive and -resistant PC3 and DU145 cells using the miRNeasy Mini Kit (Qiagen Cat# 217004). RNA-seq library construction and sequencing was performed at the Loma Linda University School of Medicine Center for Genomics. RNA-seq library was constructed using the TruSeq Stranded mRNA Low Sample Preparation protocol (Illumina; Cat# RS-1229004DOC). Two μg of total RNA were used as input. Each RNA sample was spiked with 1:100 ERCC RNA spike-in control mix 1 (Life Technologies, Cat# 4456740) prior to the first step of the protocol. All the recommended controls were used during subsequent steps including an End Repair Control, A-Tailing Control, and Ligation Control. The RNA-seq libraries were quantified using Qubit 3.0, and the quality of RNA-seq libraries was checked on Agilent TapeStation. All RNA-seq libraries were sequenced on Illumina HiSeq 4000 at the Loma Linda University Center for Genomics, with 150 bpx2, Paired-End. Quality control was confirmed (see Supplementary Figures 3 and 4).

Schistosomes were homogenized in a Bertin Minilys tissue homogenizer (MD, USA), and total RNA was isolated using Qiagen miRNeasy Mini Kit (Valencia, CA). RNA quality was assessed using an Agilent Bioanalyzer; RIN>7.5 for all samples. RNA sequencing was performed according to the manufacturer's protocol using the Illumina TruSeq RNA Sample Preparation Kit and SBS Kit v3 (San Diego, CA). Briefly, 100 ng of each total RNA sample was mixed with 2 μl of 1:1,000 diluted ERCC RNA Spike-in control Mix 1 or Mix 2 (catalogue no. 4456740/4456739, Life Technologies). This was used for polyA mRNA selection and fragmentation, followed by first and second strand synthesis, end repair, adenylation of 3′ ends and adapter ligation. Each library was enriched by 15 cycles of PCR, after which size distribution of products was validated using an Agilent Bioanalyzer and a DNA 1000 Kit. The insert size of the final library was in a band ranging from 200 to 500 bp with a peak at ∼260 bp. Libraries were quantified with a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and sequenced on a HiScanSQ System (Illumina).