ChIRP-seq (Chromatin isolation by RNA purification sequencing) experiments were performed as previously described with minor modifications [20 (link)]. In brief, seveal DNA probes against the LLNLR-299G3.1 full-length sequence were ordered from Dianxi Biotech (Shanghai, China) and were biotinylated at the 3′ end. TE1 cells were cross-linked, lysed sonicated, incubated with probe sets, and LLNLR-299G3.1-bound chromatin was retrieved. The ChIRPed RNA was purified with proteinase K buffer and then subjected for library construction using the NEBNext Ultra II RNA Library Prep kit (New England Biolabs, MA, USA). The purified DNA fragments were sequenced on an Illumina Novaseq PE150 system at Dianxi Biotech (Shanghai, China). Raw reads were mapped to human reference genome assembly (GRCh38) using the STAR (Spliced Transcripts Alignment to a Reference) version 2 software. Peaks were called using the MACS package. Motif enrichment analysis was performed using the online tool Homer (http://homer.ucsd.edu/homer/ngs/peakMotifs.html ). The sequences of probes for LLNLR-299G3.1 are listed in Suppl. Table S1.
Novaseq pe150 system
Manufactured by Illumina
Sourced in China
The NovaSeq PE150 system is a high-throughput sequencing platform developed by Illumina. It is designed to generate paired-end reads of up to 150 base pairs in length, enabling the efficient and accurate analysis of genomic samples.
Automatically generated - may contain errors
5 protocols using novaseq pe150 system
1
ChIRP-seq analysis of LLNLR-299G3.1 lncRNA
2
Chromatin Immunoprecipitation and Sequencing
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Cells were incubated in 1% formaldehyde for 10 min at room temperature, and the cross-linking process was terminated by adding glycine at the final concentration of 125 mM. The cells were lysed and then digested with micrococcal nuclease (NEB) for 20 min at 37 °C. After the cells were sonicated, the extracts were immunoprecipitated by antibodies against H3K4me3 (ab8580, Abcam) or H3K27me3 (39155, ACTIVE MOTIF) overnight at 4 °C. The extracts were then incubated in magnetic beads for 2 h at 4 °C, and the beads were washed. Finally, the DNA products were eluted and purified. ChIP-seq libraries were constructed by Novogene (Beijing, China) and sequenced with the Novaseq-PE150 system (Illumina).
3
Genomic Analysis of CRKP Strains
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To further study the CRKP strains, KP26 and KP55 in this study were selected for next-generation sequencing. Next-generation sequencing was completed by Guangzhou Weiyuan Gene Technology Co., Ltd., and the sequencing platform was a NovaSeq PE150 system from Illumina. FastQC was used for quality control of the original data, MEGAHIT software was used for genome assembly, and QUAST software was used to evaluate the quality of the genome assembly. Resistance Gene Identifier (RGI) software provided by the Comprehensive Antibiotic Research Database (CARD) was used for comparison with the CARD Database. According to the comparison results of RGI, the resistance gene information was annotated to the database. KP26 and KP55 genome circle maps were drawn by GCview software.
4
Genomic DNA Extraction and Sequencing of mcr-9-Positive Isolates
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Genomic DNA was separated from mcr-9-positive isolates with the PureLink genomic DNA minikit (Invitrogen, USA), following the instructions provided, and sequenced on the Illumina NovaSeq PE150 system by Novogene, China. The short-read data were assembled using SPAdes v3.15.4 (http://cab.spbu.ru/software/spades/ ). Identification of antibiotic resistance genes and plasmid replicon typing were conducted using the Center for Genomic Epidemiology website (http://www.genomicepidemiology.org/ ).
5
Genome Sequencing of Streptococcus suis Strain WUSS351
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A Bacterial DNA Kit (OMEGA Biotech, Norcross, GA, USA) was used to extract the genomic DNA from S. suis strain WUSS351, following the manufacturer’s guidelines. Novogene Bioinformatics Technology Co., Ltd (Beijing, China) used the Illumina NovaSeq PE150 system (Illumina, San Diego, CA, USA) to sequence the genomic DNA. SOAP denovo, version 2.04 [25 (link)] was used to carry out the de novo genome assembly. We deposited the genome sequence and annotation for strain WUSS351 at NCBI (Accession No. NZ_CP039462.1). Possible genomic islands (GIs) in the genome were predicted using Island Viewer 4 [26 (link)]. BLAST algorithms at the NCBI were used to perform homology analysis. The genetic distance among S. suis strains was calculated using the OrthoANIu algorithm [27 (link)]. Based on the distance data, a phylogenetic tree by the Neighbor-Joining method was generated with MEGA X software [28 (link)]. Multiple alignments of genomes were carried out using the Mauve software [29 (link)], and the Circos software was used to display the comparison results [30 (link)].
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