Qiaamp kit

Previously, we utilized publicly available databases to identify 382 immunomodulatory genes and these loci were used to isolate candidate SNPs of interest for genotyping^{25 (link)}. Employing resources from the MuTHER project^{28 (link),29 (link)}, 50 SNPs with the most significant cis-ieQTL activity on probes in LCLs from these immunomodulatory loci were selected for genotyping. While skin and adipose tissue data are also available in the MuTHER project, the cis-ieQTLs have been assessed from the LCL expression data, as the scope of the study is focused on the host immune cell component involved in MPM development. The selection procedure was described in detail elsewhere^{25 (link)}.
Genomic DNA from all 977 cases was isolated from whole blood samples using a QiaAmp kit (Qiagen). All SNP genotyping was completed using the MassARRAY System (Agena Bioscience Inc.) according to the manufacturer’s protocol. To ensure high-quality genotyping, quality control filters were used to remove SNPs with call rate <90%, samples with call rates <90%, and SNPs with a significant departure from Hardy-Weinberg equilibrium (p < 1E-04) resulting in 41 ieQTLs for analysis. Additionally, rare SNPs (defined by minor allele frequency <0.05) were removed prior to logistic regression analysis.

BM from WT and RIPK3^−/− donors (n=4 mice per group) was transplanted into 6-week-old RIPK3^−/− and WT recipient mice, respectively. After ablative γ-irradiation of the recipient mice, 2.5 × 10⁶ of the BM cells from donors were injected via the tail vein. After BM transfer, mice were treated with antibiotics (0.02% Borgal) for 2 weeks before the feeding experiments were started. At the end of the feeding experiment, DNA from the blood of recipient mice was extracted using the QIAamp kit (Qiagen) and analysed for confirming the success of the BM transfer.

Fecal samples from the S. marcescens outbreak group were slowly defrosted at -20°C for 24 h and 4°C for another 24 h, in order to avoid bacterial death. Portions between 0.3–0.5 g of each sample were inoculated into Brain Heart Infusion (BHI) broth (Difco, Detroit, Michigan) and incubated at 37°C for 24 h as a bacterial pre-enrichment. Cultivable bacteria were isolated in selective and nonselective agar media from the BHI tube, including agar plates of M-Enterococcus; De Man, Rogosa and Sharpe (MRS); mannitol salt; McConkey; and Columbia, with 5% sheep blood. The culture media were purchased from Difco, and the plates were incubated at 37°C for 24–48 h, including 5% CO₂ for the blood agar plates, and anaerobic conditions for the MRS plates. Colony identification was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Bruker, Germany), and all the isolates were conserved at -80°C in semi-skimmed milk. In parallel to bacterial cultures, total DNA was obtained from fecal aliquots of 0.3–0.5 g with the QiaAMP kit (Qiagen, Germany), determining their concentration and quality by Qubit fluorometer (Invitrogen, USA).

The tumors were manually dissected from 10-μm sections of formalin-fixed, paraffin-embedded tissues. Genomic DNA was extracted using the QIAamp kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) amplification of the TERT promoter region was performed using the primer pair hTERT_F (CAC CCG TCC TGC CCC TTC ACC TT)/hTERT_R (CAG CGC TGC CTG AAA CTC), generating an expected 304-bp product. The cycling conditions for PCR amplification were 95 ℃ for 2 min for denaturation, and 35 cycles of 95 ℃ denaturation for 30 s, 60 ℃ annealing for 30 s, and 72 ℃ elongation for 40 s. PCR products were confirmed by gel electrophoresis. Direct sequencing of both strands was performed using a BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) on an ABI 3500XL genetic analysis system (Applied Biosystems).

DNA was isolated from the peripheral blood using the Qiagen QIAamp kit (Qiagen, Hilden, Germany). All patients were genotyped for the PNPLA3 rs738409 c.444C>G polymorphism by the TaqMan predesigned SNP genotyping assay No. C_7241_10 (Thermo Fisher Scientific, Waltham, MA). Genotyping was performed according to the manufacturer’s protocol using the Applied Biosystems ABI 7300 Real-Time PCR instrument (Thermo Fischer Scientific). No significant deviation from the Hardy-Weinberg equilibrium was observed in PNPLA3 genotypes distribution within the CLF and HCC patient groups.