Nanodrop 1000 ultra micro spectrophotometer

Total RNA from A549 cells, A549/DDP cells, A549-exo, and A549/DDP-exo was extracted using Trizol™ reagent (15596026; Invitrogen) and Total exosomal RNA & Protein isolation kit, and the concentration of total RNA in each sample was determined using a NanoDrop-1000 ultra-micro spectrophotometer (Thermo Fisher). Reverse transcription and amplification of total RNA were performed to detect miR-424-5p expression, using Bulge-Loop miRNA qRT-PCR Starter Kit (C10211-1; Ribobio, China). TP53, SOCS5, and SOCS6 were detected using FastKing RT Kit (KR116; Tiangen, Germany) to perform reverse transcription, and Taq Pro Universal SYBR qPCR Master Mix Kit (Q712-03; Vazyme, China) to perform amplification. Primer sequences for U6, GAPDH, miR-424-5p, SOCS5, SOCS6, and TP53 are shown in Supplemental Table 2.

Genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instructions. The purity of genomic DNA was validated by NanoDrop1000 ultra microspectrophotometer (Thermo Fisher, Waltham, United States) and quantified by Qubit DNA-HS (Thermo Fisher, Waltham, United States) assay. The average genomic DNA concentration was 135ng/μl, and the final volume was 100μl. The extracted genome was sequenced on the MiSeq (Illumina, San Diego, United States) platform. Next, the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, United States) was used to construct the gene library. Afterward, the sequenced reads were assembled using the de novo mode on the CLC Genomics Workbench version 9.5.3 (Qiagen, Dusseldorf, Germany) platform (Lauraine et al., 2018 (link)). The raw sequencing data were qualified by removing 5 BP fuzzy base, bases with quality scores <20 and reads shorter than 20bp, eliminating adapter pollution, and removing repeated reads. The assembled contacts /scaffolds were first analyzed using the Rast online gene annotation analysis tool (Overbeek et al., 2013 (link)),¹ and the virulence genes were identified using VFBD (Liu et al., 2018 (link)).² Lastly, the drug resistance genes were identified using CARD (McArthur et al., 2013 (link)).³