Differential Lysis and RNA Extraction from BALF

BALF were collected from patients according to standard procedures. DNA was extracted using a QIAamp^® UCP Pathogen DNA Kit (Qiagen) following the manufacturer's instructions and 600 μL of the processed specimens was mixed with glass beads of 0.1–0.2 mm diameter. A vortex mixer (Crystal, TX, United States) was used to disrupt the bacterial cell wall at 1,600 g for 10 min. The tubes were then heated at 99°C for 10 min before DNA extraction. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma) (16 (link)). The differential lysis method was used to remove host DNA. we first use physical hypotonic lysis and chemical lysis to break human cells, and then obtain microbial cells by enzymatic hydrolysis, followed by wall breaking and nucleic acid extraction. Total RNA was extracted with a QIAamp^® Viral RNA Kit (Qiagen) and ribosomal RNA was removed by a Ribo-Zero rRNA Removal Kit (Illumina). The concentration of extracted DNA/RNA was measured using a Qubit Fluorometer before library preparation. cDNA was generated using reverse transcriptase and dNTPs (Thermo Fisher).