Dneasy blood and tissue mini kit

Genomic DNA was extracted from overnight bacterial culture using the DNeasy Blood and Tissue Mini kit (QIAGEN GmbH, Germany). Standard genomic library was prepared from the bacterial DNA and sequenced with the Illumina MiSeq platform (2 × 300 bp paired end), as described elsewhere (Klein et al., 2020 ). For quality control, raw sequences were trimmed using Sickle 1.33 (parameters, q > 30; 1 > 45). Obtained contigs were curated for length (>1000 bp) and coverage (>10×). Sequences are available under the BioProject-Numbers PRJNA561696 and PRJNA637212. Sequences were annotated using Prokka 1.14.1 (Seemann, 2014 (link)) (based on Genetic Code Table 11). Resistance genes were found using Abricate 0.8.13 with the databases from ResFinder, NCBI, CARD, ARG-ANNOT (Zankari et al., 2012 (link); Gupta et al., 2014 (link); Jia et al., 2017 (link); Feldgarden et al., 2019 (link)), to identify potential variants in the DHFR genes. For the DHPS gene, the region of interest was extracted from the assembly with Samtools (Etherington et al., 2015 (link)) and aligned with MAFFT (Nakamura et al., 2018 (link)). Unique representative sequences were obtained with CD-Hit (Li and Godzik, 2006 (link)) (100% identity) and SNPs were then called with snp-sites (Page et al., 2016 ).

Tissue samples were frozen in liquid nitrogen and stored at -80°C post resection, and subsequently transferred to RNAlater ICE^™ (Qiagen) and stored at -20°C. As described previously [23 (link)], nucleic acid extraction was performed on < 20mg of tissue by a single operator in one batch to avoid variation in protocol. Tissue disruption was carried out using a Retch Mixer mill. DNeasy Blood and Tissue Mini Kit (Qiagen) and RNEasy Plus Mini Kit (Qiagen) were used for DNA and RNA extraction, respectively. Quantification of the extracted nucleic acids was carried out using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Asheville, NC, USA) and samples were stored at -80°C.

Genomic DNA was extracted from overnight bacterial culture using the DNeasy Blood and Tissue Minikit (Qiagen GmbH, Hilden, Germany). Standard genomic library was prepared from the bacterial DNA and sequenced with the Illumina MiSeq platform (2 × 300 bp paired end), as described elsewhere [34 (link)]. For quality control, raw sequences were trimmed using Sickle 1.33 (parameters, q  >  30; 1  >  45). Clean reads were assembled with spades 3.13 with the option –careful and –only-assembler [35 (link)]. Obtained contigs were curated for length (>1000 bp) and coverage (>10×). Sequence was annotated using Prokka 1.14.1 (based on Genetic Code Table 11). Resistance genes were found using Abricate 0.8.13. Briefly, the draft genome was mapped to the database of CARD, NCBI AMRFinderPlus, Resfinder and ARG-ANNOT [36 (link),37 (link),38 (link),39 (link)] and hits with a minimum identity of 90% and a minimum coverage of 80% were considered as AMR genes present in our draft genome. The sequence was uploaded to the NCBI database under Bioproject Accession PRJNA661395.

DNA was isolated using Qiagen (Hilden, Germany) DNeasy Blood and Tissue Mini Kit. Samples were thawed and centrifuged at 16 000g for 15 minutes to precipitate DNA. After complete removal of Trizol, 180 μL buffer ATL and 20 μL protease was added and the tubes incubated at 56°C overnight before addition of 200 μL buffer AL. Samples were mixed well by vortexing before 200 μL ethanol was added and the samples were again mixed well by vortexing. The samples were then transferred to DNeasy Mini spin columns and further processed as per the manufacturer's instructions before DNA was eluted in 100 μL buffer AE. To improve recovery of the DNA, the elution buffer was left on the columns for 5 minutes before a final centrifugation step. For quantification and quality assessment of the DNA, quantitative polymerase chain reaction (qPCR) was performed with the KAPA hgDNA Quantification and QC Kit (KAPA Biosystems, Wilmington, MA) as per the manufacturer's instructions. Isolation of DNA from pure DCIS tumors were performed using the QIAcube system with the AllPrep DNA/RNA Universal Kit (cat.no. 80224, Qiagen, Hilden, Germany) according to protocol provided by the supplier.