Transcriptome profiling of cell lines from Ixodes scapularis

The goal of this study was to identify and compare baseline mRNA expression levels for a set of Ixodes scapularis cell lines originally reported in Munderloh et al. 1994 Journal of Parasitology 80(4):533-43. Three unmodified I. scapularis cell lines were included in the study: ISE6, IDE2, and ISE18. In addition, one cell population modified by insertion of a transgenic cassette using Sleeping Beauty transposition was also included: ISE18SB. The ISE18SB cells are a non-clonal cell population fully enriched for expression of a DsRed fluorescent marker gene included within the transposon insertion cassette. For all replicate samples, cells were collected after reaching approximately 60% confluency under standard culture conditions, then processed for RNA-seq analysis. Cells were cultured in airtight-capped flasks at 34 degrees C in a normoxic cell culture incubator in standard I. scapularis cell line media (Munderloh and Kurtii 1989 Experimental & Applied Acarology, 7:219-229) made in-house in our laboratory. For all replicate samples, cells were collected after reaching approximately 60% confluency (i.e., at Day 4 after seeding to fresh media). Approximately 5.5 x 10^6 cells per replicate sample were collected and processed for bulk RNA-seq analysis. All replicates were collected and processed together. RNA was extracted using Trizol and Zymo Research Direct-zol RNA Miniprep Kits. RNA was analyzed at the Biopolymers Facility at Harvard Medical School using an Agilent TapeStation as a quality control step. RIN analysis was not possible given the species; however, for all samples, the results of electrophoretic analysis profiles and quantitative PCR (qPCR) were consistent with high-quality RNA. KAPA mRNA HyperPrep Kits were used to build poly-A selected mRNA libraries. NGS was performed using an Illumina NovaSeq 6000 sequencing system with an SP flow cell. Reads were mapped to I. scapularis reference genome GCF_016920785.2 using STAR version 2.7.9a. Gene count extraction was done using FeatureCounts version 2.0.1. Normalization and conversion to FPKM values were performed using DEseq2 version 1.40.2.