2. 材料 Samples used in this study were selected from Salmonella negative piglets (crossbred or Yorkshire sows bred to boars from different breeds)
belonging to two populations of 40 and 77 individuals each and raised in climate-controlled, fully enclosed isolation facilities at the USDA-ARS-National
Animal Disease Center (NADC) in Ames, IA under identical management conditions.
サルモネラ菌感染 At 7 weeks of age, these Salmonella negative pigs received intranasal inoculation of 109 colony-forming units (cfu) of nalidixic acid
resistant Salmonella enterica serovar Typhimurium, ST _4232.
血液からのRNA調製 Approximately 2.3 ml of whole blood was collected from the jugular vein into PAXgene Blood RNA tubes (processed according to
manufacturer‘s instructions) from each individual just prior to infection (day 0) and at days 2, 7, 14, and 20 post-inoculation. Blood samples collected at day
0 and day 2 from 16 selected pigs identified as low shedding (n=8) and persistent shedding (n=8) based on extremes of Salmonella shedding levels post
innoculation were used for total RNA extraction Total RNA was prepared from 4.5-9.0 ml of solution from the PAXgene Blood RNA tubes. The DNA was
removed by in-solution DNase I digestion and RNeasy mini elute kit cleanup as recommended by QIAGEN. PCR assay without reverse transcription was
used to confirm that the RNA samples were DNA-free. The quantity and quality of the RNA were determined using Agilent 2100 Bioanalyzer (Agilent
Technologies, Santa Clara, CA) and Nanodrop 2000 (Thermo Scientific, Wilmington, DE). A globin reduction protocol developed in-house (Choi et al.;
manuscript under preperation) using porcine specific oligonucleotides was performed. Briefly, 3 ug of denatured total RNA was hybridized in a thermal
cycler at 70 degrees C for 2 min with the 10X GR oligonucleotides mix in hybridization buffer (100 mM Tris-HCL, pH 7.6; 200 mM KCl) at 70 degrees C for 5
min, then cooled to 4 degrees C. RNA-DNA hybrids were digested with 2 U RNase H (Ambion) in the reaction buffer (100 mM Tris_HCl, pH 7.6, 20 mM
MgCl2, 0.1 mM DTT, Superase-in) at 37 degrees C for 10 min and cooled to 4 degrees C. The reaction was stopped by addition of 0.5 M EDTA. The globin
depleted RNA was immediately purified with the RNeasy MinElute cleanup kit (Qiagen, Toronto, Canada, Cat. No.: 74204) according to manufacturer’s
instruction. RNA quality of the globin depleted samples was assessed using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, USA).
The mRNA library was constructed using Illumina System (TruSeq RNA sample Preparation v2 Guide, Illumina, Inc., San Diego, USA). One ug of total RNA
was purified using poly-T oligo-attached magnetic beads. The Poly (A) RNA was primed and fragmented. The first and second strand cDNA were
synthesized according to Illumina's sample preparation guide. Then individual RNA index was ligated to the end-repaired cDNA and subsequently enriched.
The quality of the mRNA libraries were confirmed using Agilent Bioanalyzer 2100 (Agilent Technologies, Inc.). The quantification was performed using
StepOne Real-Time PCR System (Applied Biosystems, Carlsbad, USA). The individual libraries were adjusted to 2 nM concentrations and pooled before
denaturation and dilution according to Illumina's instructions. The diluted libraries (8 - 10 pM) were loaded on a cBot (Illumina) for cluster generation using
the TruSeq SR Cluster Kit v3 (50 cycles, Illumina).
材料と方法
