Qiaamp dna mini qiacube kit

750 µL of saliva from each sample was transferred to a 2-mL microcentrifuge tube and mixed with 750 µl of phosphate-buffered saline (PBS). Each tube was then centrifuged for 10 min at 15,700 ×g to pellet the cells. The supernatant was discarded and resuspended the pellet in 125 μl PBS buffer and 25 μl MetaPolyzyme (Sigma-Aldrich, Norway). After incubating the tubes at 35 °C for 4 h, oral metagenomic DNA was extracted from each saliva sample by QIAcube (Qiagen, Norway) and QIAamp® DNA Mini QIAcube Kit. A pooled metagenomic DNA sample was prepared by aliquot and mixing 10 µl of each extracted oral metagenomic DNA in a new Eppendorf tube and kept at − 20 °C.

From each sample 200 µL was boiled for 10 min and DNA was extracted by QIAamp DNA Mini QIAcube kit (Qiagen, Inc., Valencia, CA, US) according to the manufacturer’s recommendations. A qPCR assay for the detection of the autolysin-encoding gene (lytA) was then performed as described before by Carvalho et al. (2007) (link). Briefly, 25 µL reaction volume composed of TaqMan Fast Universal PCR Master Mix 2x, 200 nM of each primer and probe, 10x Exo IPC-mix, 50x Exo IPC DNA and 2 µL of DNA was run at 50 °C for 2 min, denaturation at 95 °C for 10 min, followed by 40 amplification cycles of 95 °C for 15 s and 60 °C for 1 min. Samples were considered negative if cycle thresholds (Ct) exceeded 40. A positive (ATCC49619) and a non-template control (sterile water) were included in each run, along with extraction controls.

Sequences representing ituB gene homologs were extracted from all publicly available B. velezensis genomes. These sequences were aligned using CLCbio Genomics Workbench 11.0 (Qiagen Inc., Cambridge, MA, United States) and this alignment was used to identify conserved regions of nucleotides, which were used to design primers. Primers were identified for each gene cluster type (e.g., iturin, bacillomycin D, and bacillomycin L). The forward primer (ituB-fwd, 5′-CACGAACAGACAAAACA-3′) is the same for all three sets and is located on the conserved amino acid (AA) #3 module (Figure 1), while the reverse primers are located on variable AA#4 modules (Figure 1). The reverse primers are ituB-iturin-rev, 5′-TGCGCAAAGCATCGT-3′, ituB-bacD-rev5′-CTTGCGGCGTTTGTG-3′, and ituB-R-bacL5′-GGTCGCTCCTGAATCT-3′. The primers were designed to have an annealing temperature of 55 ± 2°C. PCR testing – DNA was extracted from 1 day old cultures grown on TGY for each isolate using the QIAamp DNA Mini QIAcube Kit (Qiagen Inc., Germantown, MD, United States) according to manufacturer’s protocol instructions. PCR reactions were carried out in 25 μl volumes using 2× AmpliTaq Gold master mix, 50 ng of DNA and 1 μM primers (Bvel-f and Bvel-R) under the manufacturer’s instructions (AmpliTaq Gold, Invitrogen) with an annealing temperature of 55°C.

The genomic DNA was extracted from peripheral blood by QIAamp DNA Mini QIA cube Kit (Qiagen, Hilden, Germany) before library preparation as previously described.
¹¹ (link),
²⁴ The extracted DNA was run on a 27 gene panel which was the current gene panel at URTH during the study period (approved by the Ministry of Health in Turkey). Briefly, Sophia Hereditary Cancer Solution kit (Sophia Genetics, Lausanne, Switzerland) consisting of coding regions of 27 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, FAM175A, MRE11A, NBN, PALB2, PIK3CA, RAD50, RAD51C, RAD51D, TP53, XRCC2, MLH1, MSH2, MSH6, EPCAM, PMS2, PMS2CL, MUTYH, APC, PTEN, and STK11) was used for library preparation. Sequencing was performed by NextSeq500 platform (Illumina Inc., San Diego, USA). FASTQ data were analyzed by SOPHiA Data‐Driven Medicine (Sophia DDM, Sophia Genetics v4.2, hg19 alignment). Copy numbers were identified by measuring the coverage levels of the desired regions along with samples within the same run.
²⁵ (link)

Both genomic DNA (gDNA) and total RNA were isolated from the total thyroid and thymus tissue using standard methods (QIAamp DNA Mini QIAcube Kit and RNeasy Mini Kit, QIAGEN, Hilden, Germany). An additional step of DNase I treatment was applied to the RNA samples (RNase-free DNase set kit, QIAGEN). To check for contaminating gDNA in the RNA samples, 100 ng of the total RNA were subjected to 45 cycles of PCR using specific primers for a 309 nt non-transcribed region of the CTLA4 promoter. Only samples free of DNA were used for subsequent experiments.

Genomic DNA extraction was performed manually using a QIAamp DNA mini QIAcube Kit (Qiagen, Venlo, Netherlands), according to the manufacturer’s protocol.
DNA was treated with bisulfite using an EZ-96 DNA Methylation Kit on a Zymo Spin I-96 column (Zymo Research, Irvine, CA, U.S.A.). Bisulfite-converted DNA was amplified, fragmented, and hybridized to Illumina Human Methylation 450k microarrays using an Illumina Hybridization Oven (Illumina, San Diego, CA, U.S.A.), according to the manufacturer’s protocol. Slides were analyzed by an Illumina I-Scan (Illumina, San Diego, CA, U.S.A.).
Raw iDAT files were directly imported in R software (R Development Core Team, 2008 ) and processed using the R minfi package (Aryee et al., 2014 (link)). Raw data were normalized using functional normalization (Fortin et al., 2014 (link)) before constructing the beta matrix for all 36 samples and 485,512 CpG sites (methylomics dataset).