Rna miniprep kit

We then tested the effect of patient plasma from healthy controls, febrile controls and patients with KD in U937-derived macrophages. U937 is a monocytic cell line that acquires macrophage-like properties after stimulation with phorbol 12-myristate 13-acetate (PMA, Sigma, # P8139-1) (28 (link), 29 (link)). The U937 cell line was maintained in the Roswell Park Memorial Institute culture medium (RPMI 1640, ThermoFisher) containing 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, # A1049101-01) and 1% Penicillin (GIBCO, #15140122). U937 was differentiated into macrophages by treating it with 200 μM PMA, followed by 48 h incubation in RPMI medium. Patient plasma from 10 healthy controls, 10 febrile controls and 20 patients with KD at a final concentration of 10% were then added into the medium for incubation for another 24 h. After incubation, total RNA was extracted using an RNA miniprep kit (ZYMO, #R2052). mRNA expression of CD36 and AIM2 were then determined by RT-PCR in the manner described in the previous paragraph.

Total RNA was extracted using TRIzol and purified using an RNA miniprep kit (ZymoResearch, Irvine, CA). Construction of sequencing libraries was with a TruSeq stranded mRNA library kit (Illumina, San Diego, CA). Next generation sequencing (NGS) was performed on an Illumina HiSeq 2500 system. For each sample, approximately 30 million reads were obtained. FASTQ files were aligned to the mouse reference genome using TopHat algorithm. Differentially expressed genes were analyzed using EdgerR algorithm. The related signaling pathways were viewed on the ConsensusPath database, with analyses performed using Chipster^{35 (link)}. ChIP was carried out using an Active Motif (Carlsbad, CA) kit. For input DNA, genomic DNA was sonicated to 150–800 bp using a Covaris S2 ultrasonicator (Matthews, NC). ChIP-grade TCF7 antibody was used to pull down bound DNA fragments. The Illumina library was constructed using a NEBNext Ultra II kit (Ipswich, MA), and ~ 50 million reads/sample were obtained. FASTQ files were aligned to mm10, and peaks were called using the MACS2 algorithm in Chipster. FASTQ files were uploaded to GEO.

For gene expression analysis of transporters in iBECs and hCMEC/D3, culture medium was first removed, and cells were rinsed once with D-PBS. Cells were then treated with TRIZOL ™ reagent (Thermo Fisher) and scraped off the culture plate using a pipette tip. RNA was extracted using the RNA Miniprep kit (Zymo Research) according to the manufacturer's instructions. After that, the quality and quantity of RNA was measured using a Nanodrop Spectrophotometer and RNA was converted to cDNA using SensiFAST™ cDNA synthesis kit (Bioline). 18S was used as a housekeeping gene to normalize qRT-PCR results. Following the manufacturer's instructions, cDNA samples were combined with SensiFAST™ SYBR® Lo-ROX (Bioline) and gene-specific primers to form the reaction solution. The qRT-PCR run was performed in triplicate for each sample on QuantStudio™ 5 Real-Time PCR system (Thermo Fisher Scientific). Briefly, the samples were run for 2 ​min at 95 ​°C followed by 40 cycles of 5 ​s at 95 ​°C then 30 ​s at 60 ​°C. The CT values of each gene was normalized to CT values of 18S (ΔCT values). ΔΔCt values were then calculated as 2^(−ΔCt) and multiplied by 10⁶. Multiplied ΔΔCt values were log-transformed prior to graphical presentation. Primer sequences from the genes used in this study are presented in supplementary material (Table S3).

RNA was isolated from whole cell lysate using RNA Miniprep kit (Zymo). cDNA was synthesized using random primers by MMLV reverse transcriptase (Life Technologies) with 500ng input RNA. Quantitative real-time PCR was performed using SYBR green master mix (Life Technologies). HPRT was used as a housekeeping gene. Refer to Supplemental Table 3 for primer sequences.