Truseq rna access library prep kit

Total RNA samples were extracted using an RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s recommendations. RNA quality was assessed by analysis of rRNA band integrity, using an Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA). RNA libraries were constructed using a TruSeq RNA Access Library Prep kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. The size and quality of libraries were evaluated by electrophoresis using an Agilent High Sensitivity DNA kit (Agilent Technologies); fragments were 350–450 bp. Subsequently, libraries were sequenced using an Illumina HiSeq2500 sequencer (Illumina). Sequencing data were deposited in NCBI’s Sequence Read Archive (SRA) and are accessible through BioProject accession number PRJNA516535.

Sequencing was conducted on blood for 46 patients and cultured fibroblasts for 1 patient due to sample availability. Blood-derived RNA was obtained by collecting peripheral whole blood in PAXgene blood RNA tubes and using the QIAcube system (Qiagen) according to the manufacturer’s protocol for RNA extraction. RNA was isolated from fibroblasts as previously described [34 (link)].
Sequencing libraries were prepared with either the TruSeq RNA Sample Prep Kit v2 or the TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA). Paired-end 101-basepair reads were sequenced on an Illumina HiSeq 2500 using the TruSeq Rapid SBS sequencing kit version 1 and HCS version 2.0.12.0 data collection software. A median of approximately 200 million reads was generated per individual. Base calling was performed using Illumina’s RTA version 1.17.21.3.

The total RNA from 40 matched FFPE specimens from Pre-ADT core biopsies and Post-ADT radical prostatectomies were sequenced using Illumina’s TruSeq RNA Access Library Prep kit. Sequence alignment and gene level expression quantifications were obtained using the STAR read aligner to map them to the hg38 reference genome [59 (link),60 (link),61 (link)]. Differential gene expression analysis was performed with the edgeR Bioconductor package in R. The final statistical model included corrections for sequencing the batch effects and sequence coverage. Datasets can be accessed in the NCBI GEO and SRA databases (accession No. GSE111177).

For samples that tested positive for ZIKV RNA by qRT-PCR, viral genetic diversity was characterized directly from the clinical specimens using next-generation sequencing. Sequencing libraries were prepared using the TruSeq RNA Access Library Prep kit (Illumina, Menlo Park, CA, USA) with custom ZIKV probes and sequenced using the MiSeq Reagent kit v3 (Illumina, Menlo Park, CA, USA) on an Illumina MiSeq (for details see supplementary appendix). Consensus-level ZIKV genome sequences were assembled for each sample using both de novo and reference-based approaches (for details see Supplementary Materials). These two approaches resulted in nearly identical sequences. However, for several lower coverage samples, contiguous assemblies could only be constructed with the reference-based approach. Consensus genomes were compared using median-joining haplotype networks (PopART v1.7.2) and an approximate maximum-likelihood phylogenetic reconstruction (FastTree v2.1.5). BEAST v1.8.3 [23 (link)] was used to estimate dN/dS and the rate of ZIKV evolution within the male reproductive system (MRS), HyPhy v2.3 was used to identify codons with evidence for positive, diversifying selection, and for samples with >50× average coverage, we examined intra host genetic variation using FreeBayes v1.0.2 [24 ] (for details see Supplementary Materials).

RNA was extracted using the Qiagen RNeasy FFPE kit (Germantown, MD) adhering to the manufacturer’s instructions. Library preparation was performed with the TruSeq RNA Access Library Prep Kit (Illumina, San Diego, CA). Paired-end, 75-bp reads were generated on a NextSeq 500 using a High Output, 150 cycle kit with v2 chemistry (Illumina).