Rnase free dnase digestion set

Total RNA was extracted using the RNeasy Mini RNA Isolation kit (Qiagen) according to manufacturer’s instructions. Contaminating genomic DNA was removed using the RNase-Free DNAse Digestion set (Qiagen). Total eluted RNA was reverse transcribed into cDNA using Superscript III Reverse Transcriptase and Random Primers (both from Life Technologies, Inc.). Real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems). The primer sequences for canine GAPDH, canine granzyme B, and canine perforin have been previously published (43 (link), 44 (link)). Forward and reverse primers were designed for canine granulysin (Accession XM_845424.3) using PrimerQuest software from Integrated DNA Technologies: canine granulysin (forward): 5′-TGTGTAGTGTTGCCCAGTTT-3′; canine granulysin (reverse): 5′-CTCCTTGGACACCTACTTGATG-3′. The reactions were performed on an Agilent MX3000P Real-Time PCR machine (Agilent) with the following cycling conditions: 2 min at 50°, 10 min at 95°, followed by 40 cycles of 15 s at 95° and 1 min at 60°, and a dissociation (melting) curve (15 s at 95°, 1 min at 60°). Relative gene expression was determined using the 2^−ΔΔCt method, with GAPDH as the reference housekeeping gene.

RNA was isolated from cultures using the RNeasy minikit (Qiagen) and its associated RNase-free DNase digestion set (Qiagen), in accordance with the manufacturer’s protocol for mammalian cells. An Agilent bioanalyzer was used to check the quality of the RNA samples. Tru-Seq stranded mRNA libraries were generated from 5 to 17 ng/μl of mRNA for THP-1 cells, and from 50 to 120 ng/μl of mRNA for murine peritoneal exudate cells (PECs) and placental explants, and sequenced with an Illumina NextSeq 500 sequencer. mRNA-Seq FASTQ reads were mapped to the human reference genome (Homo sapiens v81; hg38) using default options on CLC Genomics Workbench 11 (Qiagen). Total gene reads (with at least 1 read count) were exported from CLC Genomics Workbench and used for DESeq2 (56 (link)) to perform differential expression analysis using methods outlined previously (for an example, see reference 4 (link)). Data were evaluated using principal-component analysis (embedded in the DESeq2 package), and genes were deemed to be significantly expressed if the log₂ fold change was greater than or equal to 1 or less than or equal to −1 and with an adjusted P value (P_adj) value of <0.01. Gene set enrichment analysis (GSEA) (57 (link)) and Ingenuity pathway analysis (Qiagen) (58 (link)) software were used to compare gene sets that were differentially regulated after infection with WT and ΔGRA28 parasites.