Tianamp magnetic dna kit

BALF samples were collected from patients according to standard procedures. The TIANamp Magnetic DNA Kit (Tiangen) was used to extract DNA. The quantity and quality of DNA were, respectively, assessed using the Qubit (Thermo Fisher Scientific) and NanoDrop (Thermo Fisher Scientific).

DNA extraction from the BALF was performed as described in [17 (link)]. Briefly, 1 ml BALF was digested with 50 μl protease K at 60°C for 20 min and then placed at 4°C for 5 min. The sample was transferred into a sterile 5 ml tube, followed by brief centrifugation, and the DNA was extracted using the TIANamp Magnetic DNA kit (DP710-t2, Tiangen, China), according to the manufacturer's protocol. A no-template control (NTC) was performed for PCR. The quantity was assessed using the Qubit 2.0 fluorometer (Thermo Fisher Scientific, USA), and the quality of DNA was evaluated using the Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, USA). BALF DNA was fragmented into 150–300 bp size range by using the Bioruptor Pico Plus (Diagenode, Belgium) with the ultrasonication parameters as follows: 30 s on, 30 s off; 10 cycles. The DNA library was constructed using the KAPA HyperPrep kit (KAPA Biosystems, USA), according to the manufacturer's protocol. The library was qualified with Agilent 2100 (Agilent Technologies, CA) and sequenced on Illumina NextSeq 550Dx (Illumina, USA) using 75 bp single-ends.

BALF DNA was extracted using methods previously described (Mac Aogáin et al., 2021 (link); Ju et al., 2022 (link)), take 50 μL of proteinase k and 1 mL of BALF sample, digest at 60°C for 20 min, and then leave at 4°C for 5 min to lower the reaction temperature. Transfer the sample to a sterile test tube and centrifuge briefly followed by DNA extraction using the TIANamp Magnetic DNA Kit (DP710-t2, Tiangen, China) according to the manufacturer’s protocol. Sputum was liquefied by 0.1% DTT (dithiothreitol) for 20 min at 56°C before extraction. The QIAamp Viral RNA Mini Kit (Qiagen) was used to extract RNA from the BALF (Langelier et al., 2018 (link)).
DNA libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer’s protocol. Libraries were constructed after Qubit quantification. For RNA extraction samples, rRNA was removed from total RNA and libraries were constructed after purification as described for DNA library construction. Agilent 2100 was used for quality control and then DNA libraries were sequenced on the Dif seq platform for 50 bp paired end sequencing (Dinfectome Medical Technology Inc, Nanjing, China).