Nextseq cn500 sequencer

All samples were extracted with QIAamp^® UCP Pathogen DNA Kit (Qiagen). Tween 20 and Benzonase (Qiagen) are used to remove DNA from human samples according to the manufacturer’s instructions (Amar et al., 2021 (link)). A QIAamp^® Viral RNA Kit was used to extract RNA, and a Ribo-Zero rRNA Removal Kit (Illumina) was used to remove ribosomal RNA. cDNA was synthesized with reverse transcriptase and dNTP (Thermo Fisher). The construction of the selected DNA and cDNA library was done using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) (Miller et al., 2019 (link)). Quality control for the library was performed using a Qubit dsDNA HS Assay Kit and a High Sensitivity DNA kit (Agilent) on an Agilent Bioanalyzer 2100. Then, the library pools were loaded onto an Illumina NextSeq CN500 sequencer for 75 cycles of single-end sequencing of 20 million reads for each library. Negative control (NC) is a peripheral blood mononuclear cell sample of 105 cells/mL prepared alongside each batch with the same protocol (Amar et al., 2021 (link)), and sterile deionized water was used as a nontemplate control (NTC) alongside the specimens (Li et al., 2018 (link)).

The conventional culture methods include bacterial culture, fungal culture, acid-fast bacterial culture, and blood culture. Blood cultures contain aerobic and anaerobic cultures. We performed conventional culture and mNGS methods according to the patient’s medical conditions, and finally selected cases with the same specimens for examination to be included in this study. However, it was not required that all types of cultures be performed on each patient. Microbial culture and automated microbial identification systems were utilized.
Following standard operating procedures, nucleic acid extraction, nucleic acid fragmentation, end repair, end adenylation, primer ligation, and purification were performed from each sample to form a sequencing library using kits from an automated workstation (Amar et al., 2021 (link)). Libraries were assessed for quality using kits quantified by real-time PCR and loaded onto an illumina Nextseq CN500 sequencer for 75 cycles of single-end sequencing, producing approximately 20 million reads per library (Miller et al., 2019 (link)). Furthermore, peripheral hematopoietic cell specimens from healthy donors were used as negative controls simultaneously, and sterile deionized water was represented as non-template controls concurrently with each batch (Miller et al., 2019 (link)).

The peripheral blood samples of 3 ATC patients and 3 normal healthy persons in The Second Hospital of the University of South China were collected, and total RNA was isolated by TRIzol reagent (Invitrogen). NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) was applied to measure RNA sample concentration as the OD260/280 ratio. The integrity of RNA was checked by agarose gel electrophoresis with 28S: 18S ratio ≥ 1.5. circRNA sequencing was performed using the Illumina NEBNext Ultra RNA Library Prep Kit (NEB, USA). An Illumina TruSeq® Small RNA Library Prep Kit was used as the miRNA sequencing library. A total of 5 μg RNA was used for each sample. RNA sequencing library quantification kit (KAPA Biosystems) was applied for quantification and quality inspection. Paired-end sequencing was completed on the Illumina NextSeqCN500 sequencer. circRNA sequencing was performed on the peripheral blood samples of 3 ATC patients and 3 normal healthy people to screen the differentially expressed circRNAs in the peripheral blood samples of ATC patients.

DNA was extracted from all BALF samples using a QIAamp^® UCP Pathogen DNA Kit (Qiagen). Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma). Total RNA was extracted with a QIAamp^® Viral RNA Kit (Qiagen), and ribosomal RNA was removed by a Ribo-Zero rRNA Removal Kit (Illumina). cDNA was generated using reverse transcriptase and dNTPs (Thermo Fisher). Libraries were constructed for the DNA and cDNA samples using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA). Library was quality-assessed by Qubit dsDNA HS Assay kit followed by a high-sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq CN500 sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library.

Bronchoscopy was performed according to standard procedures using a flexible fiberoptic bronchoscope. A special collector was used to collect 3–5 ml of BALF, which was stored at 4°C. The BALF was sent for mNGS analysis (DNA and RNA). DNA was extracted using a QIAamp^® UCP Pathogen DNA Kit (Qiagen), following the manufacturer’s instructions. Human DNA was removed using benzonase (Qiagen) and Tween20 (Sigma). Total RNA was extracted using a QIAamp^® Viral RNA Kit (Qiagen). Ribosomal RNA was removed using a Ribo-Zero rRNA Removal Kit (Illumina). Complementary DNA (cDNA) was generated using reverse transcriptase and deoxynucleoside triphosphates (Thermo Fisher Scientific). Libraries were constructed for DNA and cDNA samples using the Nextera XT DNA Library Prep Kit (Illumina). Library quality was assessed using the Qubit dsDNA HS Assay Kit, followed by a high-sensitivity DNA Kit (Agilent) on an Agilent 2100 bioanalyzer. Library pools were then loaded onto an Illumina NextSeq CN500 sequencer for 75 cycles of single-end sequencing, generating approximately 20 million reads per library. For negative controls, we prepared peripheral blood mononuclear cell samples (10⁵ cells/ml) from healthy donors, in parallel with each batch, using the same protocol. Sterile deionized water was extracted alongside the specimens to serve as a non-template control.

A total of five pairs of eutopic and ectopic endometrial tissues of O-EMs patients were subjected to the extraction of total RNA following the protocols of Trizol reagent (Invitrogen, Carlsbad, CA). The concentration of the RNA was then determined by Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, San Jose, CA) with the optical density (OD) measured at 260/280 wavelength, and the integrity of RNA was detected by agarose gel electrophoresis (28 s/18 s ratio >1.5). After that, the An Illumina TruSeq^® Small RNA Library Prep Kit was utilized for miRNA sequencing, and a total of 5 µg RNA from each sample was detected, followed by quantification and quality control using the KAPA Library Quantitative Kit (KAPA Biosystems, Woburn, MA). Then, paired-end sequencing was performed on the Illumina NextSeqCN500 sequencer, and differentially expressed miRNAs related to O-EMs were screened out through the high-throughput transcriptome sequencing of the eutopic and ectopic endometrial tissue samples from O-EMs patients.