title: GSM4365432: 1008: Female Kidney, Hank's Balanced Salt Solution; Pimephales promelas; RNA-Seq
library_construction_protocol: Kindey was removed approximately 8 hours following pathogen injection, placed on dry ice and stored at -80°C until RNA extraction. RNA was extracted using the Promega Maxwell 16 LEV simplyRNA Purification Kit per manufacturer protocol. To prepare cDNA libraries, the NEBNext Ultra II RNA Library Prep kit in conjunction with the NEBNext poly(A) mRNA magnetic isolation module and NEBNext Multiplex Oligos for Illumina (New England BioLabs, Ipswich, MA) were utilized according to manufacturer protocols with an input of 500ng total RNA. Library quality (e.g. fragment size, presence of adaptors) was assessed using the Experion DNA 1K Analysis kit in conjunction with the Experion Automated Electrophoresis System (BioRad, Hercules, CA). Finally, libraries were quantified using the NEBNext Library Quant Kit (BioRad, Hercules, CA) and shipped to University of Texas Health Science Center San Antonio where paired end sequencing (100bp) was performed on the Illumina HiSeq 3000 platform. Adaptor sequences and low-quality reads were removed from raw sequences using Trimmomatic v0.38 (Bolger et al. 2014). Specifically, trimming was applied using a sliding window approach with a minimum quality score of 20 followed by a minimum length filter of 36bp as recommended by Williams et al. (2016) and Bolger et al. (2014). Sequence quality was assessed before and after trimming using FastQC. Two samples from each treatment group containing the greatest number quality filtered of reads were combined, and a de novo assembly was generated with Trinity v2.4.0 using default settings (Haas et al. 2013).  The de novo assembly was then annotated against the UniProtKB Swiss-Prot (www.uniprot.org) database using BLASTX with a cutoff E-value of less than 1.0 x 10-5. Individual samples were aligned to the de novo assembly using RNAseq by Expectation Maximization (RSEM v.1.3; Li & Dewey, 2011) to obtain gene-level counts.