Novaseq sequencing

To investigate the selection on ESBL production, DNA was isolated from 1 ml of feces and 1 ml of manure slurry of all nine tubes after experiment 2 and 3, using the protocol as described for the EFFORT-project⁵³ . Illumina sequencing was performed as described by the manufacturer using 150 bp paired end Illumina Novaseq sequencing Libraries were created using the Ilumina Nextera XT DNA Library Preparation Kit according to the manufacturer’s protocol⁵⁴ . Resulting DNA reads were processed using FastDeME according to the default settings⁵⁵ . Reads were processed using FastP to remove poor quality reads, sequencing adapters and barcodes^{56 (link)}. To investigate the resistome, Kaiju was used to profile the reads for bacterial species identification and the tool KMA was used to detect allignments to known antimicrobial resistance genes with the default Resfinder database^{57 (link),58 (link)}. The sequence depth of each resistance gene was adjusted for sequencing depth to depth per gigabase. Resistance classes were obtained from the Resfinder database⁵⁹ and hits to each resistance gene were summed per antimicrobial class. Sequence data is available under accession PRJEB49007 at the sequence read archive (SRA). Results were visualized using Microsoft Excel.

According to the manufacturer’s protocol, DNA was extracted from the soil samples using the E.Z.N.A.^® Soil DNA Kit (D5625, Omega, Inc., USA). The 341F-805R (5′-CCTACGGGNGGCWGCAG-3′/5′-GACTACHVGGGTATCTAATCC-3′) and ITS1FI2-ITS2 (5′-GAACCWGCGGARGGATCA-3′/ 5′-GCTGCGTTCTTCATCGATGC-3′) primer sets were used to amplify the bacterial 16S V3-V4 region and fungal ITS2 genes. Amplicon synthesis, library construction, and Illumina NovaSeq sequencing (2 × 250 bp) were performed by LC-Bio Technology Co., Ltd. (Hangzhou, China). FLASH (Magoc and Salzberg, 2011 (link)) was used to construct paired-end 16s and ITS1 sequences, which were subsequently quality-trimmed and length-filtered using Fqtrim. DADA2 (Callahan et al., 2016 (link)) was used to construct the amplicon sequence variant (ASV) table, which was then allocated to the proper taxon using the QIIME 2 plugin (Bolyen et al., 2019 (link)). Taxonomy was assigned against the SILVA (release 132, https://www.arb-silva.de/documentation/release-132/) (Quast et al., 2012 (link)) and Unite (V8 released on 02.02.2019) databases (Abarenkov et al., 2010 (link)). The samples were rarefied to 51,038 sequences for bacterial communities and 38,330 sequences for fungal communities.

The OMEGA Soil DNA Kit (M5635‐02) (Omega Bio‐Tek) was used to extract the genomic DNA of stool samples, the DNA was quantified by Nanodrop, and the quality of DNA extraction was detected by 1.2% agarose gel electrophoresis. Based on the 16S rRNA V3–V4 region of microorganisms, the corresponding primers were designed: F: ACTCCTACGGGAGGCAGCA; R: GGACTACHVGGGTWTCTAAT, and the sample‐specific Barcode sequence was added, and then the rRNA gene V3–V4 fragment was amplified by PCR. The amplification products were purified and recovered by magnetic beads, and the PCR amplification products were quantified by fluorescence. The fluorescent reagent was Quant‐iT PicoGreen dsDNA Assay Kit, and the quantitative instrument was Microplate reader (BioTek, FLx800). According to the fluorescence quantitative results, each sample is mixed in a corresponding proportion according to the sequencing volume requirement of each sample. Illumina NovaSeq sequencing was used to prepare the sequencing library using Illumina's TruSeq Nano DNA LT Library Prep Kit.

Total RNA was extracted from 100 mg of barley leaves infected with Fg using the RNA isolation kit (Zymo-R2072, Irvine, CA, USA), RNA extraction buffer (50 mM Tris-HCl pH = 8.0, 150 mM LiCl, 5 mM EDTA pH = 8.0, 1% SDS), Trizol reagent, and phenol: chloroform (1:1), 80% ethanol) according to the manufacturer’s protocol. The isolated RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies^®, Wilmington, DE, USA). Quality was assessed using 1% agarose gel and running on BioAnalyzer 2100 (Agilent Technologies^®, Santa Clara, CA, USA). Samples having 260/A280 > 2.0, RIN > 8, and concentration >50 ng/µL were selected for downstream applications. Approximately 2 μg of high-quality RNA was sent to a biotech company (GENEWIZ^®, Leipzig, Germany) to prepare the Illumina standard RNA library with polyA selection. The company performed Illumina NovaSeq sequencing with an estimated data output of ~20 M paired-end reads with a quality score of Q30. The company provided FASTQ format data that were used for further analysis.

Male hippocampi were dissected and total RNA was extracted using Trizol reagent (Invitrogen). mRNAs were purified using poly-T oligo-attached beads and fragmented for cDNA synthesis. cDNAs were sequentially synthesized and a single ‘A’ nucleotide is added to 3′ end of cDNAs. Multiple indexing adapters are ligated to 5′ and 3′ of the ends of the cDNAs. A library was constructed and validated on the Agilent 2,100 Bio-analyzer and Real-Time PCR System. Subsequently, Illumina NovaSeq sequencing was proceeded. The RNA-seq reads were trimmed to remove the adaptor and low-quality sequences by using Trimmomatic. The trimmed reads were mapped to the mouse reference genome sequence (mm10) using Bowtie2. Quantification of gene expression was performed by EBseq. The Heatmap was generated by online ClustVis (https://biit.cs.ut.ee/clustvis/). Gene set enrichment analysis was performed to provide more information about the biological functions and pathways significantly enriched in up- or down-regulated genes (DEGs) by focusing on gene ontology (GO) term (BP, biological process) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using Cluster Profiler.

For transcriptomic analysis, BMSCs were treated with 2 μg/ml ESPs, HCF, or pLL for 48 h. Total RNA was subsequently extracted with TRIzol, quality-assessed, and used to construct cDNA libraries for Illumina NovaSeq sequencing. Differential expression analysis was conducted using Deseq2.

Zebrafish retinas (4–6 retinas from 4–6 fish) were collected at different time points after NMDA and LD treatments, and flash-frozen in dry ice for ~15 min before being transferred to a −80 °C freezer for storage. Nuclei were extracted from frozen retinal tissues according to 10xMultiome ATAC + Gene Expression (GEX) protocol (CGOOO338). Briefly, frozen retinal tissues were lysed in ice-cold 500 ml of 0.1X Lysis buffer using a pestle and incubated on ice for 6 min totally. Nuclei were centrifuged, washed 3 times and resuspended in 10xMultiome nuclei buffer at a concentration of ~3000–5000 nuclei/ml. Nuclei (~15k) then were loaded onto 10x Genomic Chromium Controller, with a target number of ~10 K nuclei per sample. RNA and ATAC libraries were prepared according to the 10xMultiome ATAC + Gene Expression protocol and subjected for Illumina NovaSeq sequencing at ~500 million reads per library.