The plant growth-promoting rhizobacteria (PGPR) Bacillus velezensis strain WRN014 was isolated from banana fields located in Hainan Province in China. The strain was grown in Luria-Bertani (LB) broth at 30°C with moderate shaking and the cell was used for genomic extraction. The complete genome of Bacillus velezensis WRN014 was sequenced by combining Illumina Hiseq 2500 system and PacBio RSII high-throughput sequencing technology. The reads of the Illumina Hiseq 2500 system were assembled using an assembly pipeline called A5 (Andrew and Aaron's Awesome Assembly Pipeline) [24] and the software SPAdes genome assembler [25] . The reads of PacBio RSII were assembled into contigs using Hierarchical Genome Assembly Process 4 (HGAP4) [26] and Canu [27] . The gaps between contigs were filled by comparing the contigs that were assembled from the Illumina Hiseq 2500 system and PacBio RSII using the software MUMmer [28] . The quality of genome assembly was improved using the software Pilon [29] . The complete genome of WRN014 was annotated using the Prokaryotic Genomes Annotation Pipeline (PGAP) at NCBI [30] .
Hiseq 2500 system
Manufactured by Illumina
Sourced in United States, China, United Kingdom, Japan, Sweden, Germany, Canada, Italy, India, Spain, Switzerland, Cameroon
The HiSeq 2500 system is a high-throughput DNA sequencing platform developed by Illumina. It is designed to perform rapid, high-quality sequencing of DNA samples. The HiSeq 2500 system utilizes Illumina's proprietary sequencing-by-synthesis technology to generate sequencing data.
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Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (123 mentions)
Trizol reagent, by Thermo Fisher Scientific (94 mentions)
Rneasy mini kit, by Qiagen (51 mentions)
Trizol, by Thermo Fisher Scientific (41 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (36 mentions)
2100 bioanalyzer, by Agilent Technologies (36 mentions)
Truseq rna sample preparation kit, by Illumina (25 mentions)
Nanodrop, by Thermo Fisher Scientific (24 mentions)
Bioanalyzer, by Agilent Technologies (21 mentions)
Rneasy kit, by Qiagen (20 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (18 mentions)
Ampure xp bead, by Beckman Coulter (17 mentions)
Agencourt ampure xp bead, by Beckman Coulter (16 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (16 mentions)
Rneasy micro kit, by Qiagen (16 mentions)
Mirneasy mini kit, by Qiagen (16 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (16 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (15 mentions)
Rneasy plant mini kit, by Qiagen (15 mentions)
Truseq rna sample preparation kit v2, by Illumina (15 mentions)
1 300 protocols using hiseq 2500 system
1
Genome Sequencing of Bacillus velezensis WRN014
2
Transposon Sequencing for Bacterial Mutants
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For Azoarcus olearius DQS4T, a bacterial culture at mid-log phase grown at 37°C was collected for DNA extraction. Cells were pelleted by centrifugation and immediately frozen at −80°C until DNA extraction. Genomic DNA was extracted, and transposon insertion junctions were selectively amplified, as described previously (79 (link)). Insertion junction libraries were multiplexed and sequenced using the Illumina HiSeq 2500 system. For Herbaspirillum seropedicae SmR1, libraries were sequenced on either the HiSeq 2000 or HiSeq 2500 system (Illumina) to map a greater fraction of the mutant population. Genomic DNA was extracted, and barcodes were amplified as described previously (16 (link), 18 (link), 80 (link)).
3
Transcriptomic Analysis of TP-472 Treatment in A375 Cells
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A375 cells were treated with vehicle control (DMSO) or 5 μM or 10 μM TP-472 for 24 h, after which total RNA was extracted for analysis of gene expression on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions and purified using RNAeasy mini columns (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Finally, mRNA was purified from approximately 500 ng of total RNA using oligo-dT beads and sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific libraries. The resulting cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using uracil-DNA-glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert-size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μL were sequenced on an Illumina HiSeq 2500 system. Images were converted into nucleotide sequences using the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.
4
RNA-Seq of Ovarian Cancer Cell Lines
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Ovarian cancer cell lines (PA-1 and SK-OV3 cells) were treated with BAY-850 (5 μM) and control for 48 h were used to prepare total RNA for gene-expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions and purified on RNAeasy mini columns (Qiagen) according to the manufacturer’s instructions. Then, mRNA was purified from approximately 300 ng total RNA using oligo-dT beads and sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert-size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥ 0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.
5
Genomic Analysis of PAS-Resistant Strains
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We collected eight PAS-resistant clinical strains from the Chongqing Public Health Medical Center [19 (link)]. Genomic DNA of the clinical isolates was extracted using a HiPure mycobacteria DNA kit (Magen, China) and sent to Shanghai Biotechnology Corporation for whole-genome sequencing. The library was constructed using the NEBNext Ultra DNA library prep kit for Illumina (NEB, USA) and sequenced on a HiSeq2500 system (Illumina, USA). The library concentration and size were measured using a Qubit® 2.0 fluorometer and Agilent high-sensitivity DNA kit (Agilent, USA), respectively. Then, the library was sequenced using a HiSeq 2500 system (Illumina, USA). All experimental steps were carried out following the manufacturer’s protocol. All data of the whole genome sequencing of the eight PAS-resistant clinical strains were uploaded as accession no. PRJNA774929 [20 ]. Raw sequence data were cleaned and then mapped to the H37Rv reference genome (GenBank accession number AL123456.3) by BWA software (version 0.7.12). Single nucleotide variants and indels were identified by the GATK tool, and structural variations were identified by the FermiKit tool.
6
Transcriptome and Genome Sequencing Protocols
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Using 1-ng aliquots of each cDNA sample, a WTS library was prepared using a Nextera XT DNA Sample Prep Kit (Illumina), according to the manufacturer's instructions. Then, the libraries were sequenced on a HiSeq 2500 system using 100-bp paired-end sequencing.
WGS libraries were constructed using a TruSeq Nano DNA Library Prep Kit (Illumina) according to the protocol for sample preparation for multiplexed paired-end sequencing. Low-coverage genome sequencing was performed on an Illumina HiSeq 2500 system with 100-bp paired-end sequencing.
Sequencing libraries for WES were created using the SureSelect XT Human All Exon V5 kit (Agilent Technologies) and subsequently analyzed by the HiSeq 2500 systems (Illumina) using the 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina).
WGS libraries were constructed using a TruSeq Nano DNA Library Prep Kit (Illumina) according to the protocol for sample preparation for multiplexed paired-end sequencing. Low-coverage genome sequencing was performed on an Illumina HiSeq 2500 system with 100-bp paired-end sequencing.
Sequencing libraries for WES were created using the SureSelect XT Human All Exon V5 kit (Agilent Technologies) and subsequently analyzed by the HiSeq 2500 systems (Illumina) using the 100-bp paired-end mode of the TruSeq Rapid PE Cluster kit and TruSeq Rapid SBS kit (Illumina).
7
RNA-seq Analysis of EPZ-5676 Treatment
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IGROV-1 and SK-OV-3 cells treated with vehicle or 10 µM EPZ-5676 for 48 h were used to prepare total RNA, which was then used for gene expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen) according to the manufacturer’s instructions and then purified on RNAeasy mini columns (Qiagen) according to the manufacturer’s instructions. mRNA was purified from ~500 ng total RNA using oligo-dT beads and then sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific sequencing libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images generated by the sequencers were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.
8
RNA-seq of Aging Transcriptome
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RNA was extracted using a Trizol/RNeasy hybrid protocol. In brief, after phase separation, the aqueous phase was mixed with one volume of 70% ethanol and passed through an RNeasy spin column (QIAGEN RNeasy Mini Kit). RNA quality was determined using the high-sensitivity RNA ScreenTape assay (Agilent 5067-5579), and all samples had RIN scores >7.0. RNA-seq libraries for differential expression or splicing analysis were prepared for four biological replicates/conditions, including four young adult males, four aged males, four young adult females, and four aged females per library type. For differential expression analysis, total RNA libraries were prepared using the TruSeq Stranded Total RNA library prep kit with Ribo-Zero (Illumina 20020596). Paired-end 100 bp sequencing to a depth of ~50 million reads per sample was performed using the Illumina HiSeq 2500 system at the UCLA Broad Stem Cell Research Center Sequencing Core. For splicing analysis, mRNA libraries were prepared with poly-A selection using the TruSeq Stranded mRNA library prep kit (Illumina 20020594), and paired-end 75 bp sequencing to a depth of ~50 million reads per sample was performed using the Illumina HiSeq 2500 system at the UCLA Neuroscience Genomics Core.
9
Transcriptomic Analysis of UBE2T Knockdown
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MCF7 and T47D cells expressing NS or UBE2T shRNA were used to prepare total RNA, which was then used for gene expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol® reagent (Invitrogen), according to the manufacturer's instructions, and purified on RNeasy mini columns (Qiagen), according to the manufacturer's instructions. mRNA was purified from ∼500 ng total RNA using oligo-dT beads and sheared by incubation at 94°C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific sequencing libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using uracil–DNA–glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qPCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images generated by the sequencers were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in fastq format.
10
Transcriptome Profiling of Plant Root Samples
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Total RNA was extracted from six root samples (CK-1, CK-2, CK-3, T-1, T-2 and T-3) following the manufacturer’s instructions of Trizol reagent (Invitrogen, Carlsbad, CA, USA). The purity and quality of total RNA were measured on Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA) and Qubit® 2.0 (Invitrogen, Life Technologies, CA, USA). Following the protocol of the Gene Expression Sample Prep Kit (Illumina, San Diego, CA, USA), six libraries (CK-1, CK-2, CK-3, T-1, T-2 and T-3) were constructed. These six libraries were sequenced on an Illumina HiSeqTM 2500 system with the pair-end and 125 bp mode by BIOMARKER (Beijing, China). Raw sequence data were deposited in Short Read Archive (SRA) of National Centre for Biotechnology (NCBI) and are available under BioProject accession PRJNA326331.
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