Genomic DNA (gDNA) was extracted from faecal and mucosal samples by using QIAamp® DNA Stool Mini Kit (Qiagen, Courtabœuf, France) and NucleoSpin® Tissue Kit (Macherey-Nagel, Hoerdt, France), respectively, following the manufacturer’s instructions. Five mice per group were chosen for 16S rRNA gene analysis by high-throughput sequencing (Illumina) based on both the histological score and the DNA quantity and quality. The V4 variable region of the 16S rRNA genes was amplified by PCR using universal primers F515 (5′-GTGCCAGCMGCCGCGGTAA-3′) and R806 (5′-GGACTACHVGGGTWTCTAAT-3′)54 (link). The DNA library was constructed following TruSeq DNA library preparation protocol (Illumina, San Diego, CA, USA). Paired-end sequencing (2 × 250 bp) was performed at MR DNA (www.mrdnalab.com , Shallowater, TX, USA) on a MiSeq platform.
High throughput sequencing
Manufactured by Illumina
Sourced in China, United States, United Kingdom
High-throughput sequencing is a laboratory equipment used for rapid and efficient DNA or RNA sequencing. It enables the simultaneous analysis of multiple genetic samples, allowing for the generation of a large amount of sequencing data in a relatively short time.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (4 mentions)
Nextseq, by Illumina (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Dneasy powersoil kit, by Qiagen (2 mentions)
Truseq dna library preparation protocol, by Illumina (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Hiseq 2000, by Roche (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
Ovation rna seq system, by Tecan (1 mentions)
Stool genomic dna kit, by CoWin Biotech (1 mentions)
Axyprep dna gel recovery kit, by Corning (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Q5 high fidelity dna polymerase, by Takara Bio (1 mentions)
Pcr fragment purification kit, by Takara Bio (1 mentions)
Hiseq2500 pe250, by Illumina (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Rt122, by Tiangen Biotech (1 mentions)
Dp304, by Tiangen Biotech (1 mentions)
50 protocols using high throughput sequencing
1
16S rRNA Microbiome Analysis of Faecal and Mucosal Samples
2
High-throughput sequencing of CRISPR gRNA
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To amplify the gRNA fragments integrated in the genomic DNA for Illumina high-throughput sequencing, we performed two-step PCR amplification using primers flanking the gRNA protospacers. For each sequencing library, 16ug of genomic DNA was used. PCR1 was performed using the primers with stagger barcodes to maintain sequence diversity across the flow-cell. PCR2 was performed to add full-length Illumina sequencing adapters using custom primers with index for multiplexing. All PCRs were performed using NEBNext polymerase (New England Bioscience). To avoid overamplification, between PCR1 and PCR2, we performed qPCR to gauge how much product we have and calculate the number of cycles of PCR2 to be performed. The pooled samples were sequenced using NextSeq (Illumina) at the Utrecht Sequencing Facility.
All primer sequences are listed inTable S3 .
All primer sequences are listed in
3
Genome-wide Mapping of Su(Hw) Binding Sites
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Genome-wide association of Su(Hw)M4M393 was determined using ChIP, using ∼200 ovary pairs dissected from su(Hw)M393/v females younger than 6 h old per experiment, as described previously (31 ). Single-end libraries for Illumina high-throughput sequencing were prepared from ∼100 ng of DNA from each fraction (Cincinnati Children's Hospital Medical Center Genetic Variation and Gene Discovery Core Facility, Cincinnati, OH, USA). Illumina Genome Analyzer IIx fastq files were processed as described previously (31 ). ChIP-seq datasets were evaluated using Partek v. 6.5. The Su(Hw)M4M393 ChIP-seq1 generated over 32 million mapped reads for anti-Su(Hw) IP and over 34 million mapped reads for the control pre-immune IP. A replicate experiment, Su(Hw)M4M393 ChIP-seq2, generated over 40 million mapped reads for anti-Su(Hw) IP and over 47 million mapped reads for the control pre-immune IP. ChIP-seq1 identified 636 sites using a 1% false discovery rate (FDR) and a 3× fold-enrichment (IP versus IgG) cutoff, and ChIP-seq2 recovered 329 sites using a 1% FDR and a 1.5× fold-enrichment (IP versus IgG) cutoff. Over 90% of SBSs identified in ChIP-seq2 were identified in Su(Hw)M4M393 ChIP-seq1 (Supplementary Figure S2 ). ChIP-seq data are submitted to the NIH GEO/Sequence Read Archive database, accession number GSE86243.
4
Illumina and 454 Sequencing of Bacterial 16S rRNA
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The obtained genomic DNA was then submitted to the Illumina high-throughput sequencing (samples S1 and S7) and 454 pyrosequencing (samples S1–S8), which were performed using the Illumina Hiseq 2000 and FLX Titanium platform of Roche 454 from the Beijing Genome Institute (Shenzhen, China), respectively. The bacterial 16S rRNA gene (V3-V4 region, approximately 460 bp) was carried out with Illumina-specific fusion primers V3F and V4R (Claesson et al., 2009 (link)). PCR amplification was conducted with previous report (Huang et al., 2014 (link)). The 12-bp barcode with primers was used to assign individual sequences to samples. The 16S rRNA gene amplicons were submitted to an Agilent 2100 Bioanalyzer (Agilnet, United States) before using FLX Titanium platform of 454 pyrosequencing (Roche, United States). The GenBank accession numbers for the genomic datasets in NCBI are SRX825942 and SRX825518 .
5
Transposon Sequencing for Tunicamycin Response
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Transposon sequencing was performed as described previously42 (link). Briefly, a pooled transposon library was grown in the presence or absence of 1 μg/ml tunicamycin at 30 °C for 5 h. Genomic DNA was then harvested and prepared for Illumina high-throughput sequencing for identification of the genomic location of the transposon in the surviving mutant. Sequencing data was processed using the Tufts University Genomics Core Facility Galaxy server43 (link)–45 (link) and mapped to the genome of NCTC832 using Bowtie software46 (link). Mapped reads were processed to identify depleted genes using the Mann-Whitney U test. Genes that had a >5-fold change in the number of reads between control and treated samples were considered enriched or depleted, and the significance threshold was set to P < 0.05 after correcting for false discovery rate.
All genes that were considered a hit in any comparison were then compiled. The depletion ratio for each gene in each sample was then combined by geometric averaging (taking the sixth root of the product of all six depletion ratios). P values were combined by using the Chi Squared statistic with six degrees of freedom.
All genes that were considered a hit in any comparison were then compiled. The depletion ratio for each gene in each sample was then combined by geometric averaging (taking the sixth root of the product of all six depletion ratios). P values were combined by using the Chi Squared statistic with six degrees of freedom.
6
Filamentous Bulking Sludge Analysis in Chinese WWTPs
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Activated sludge samples were collected from the aeration tanks of four WWTPs located in the north of Xinjiang Uygur Autonomous Region of China in winter. These four WWTPs applied oxidation ditch process. The following 8 samples were collected: ALT1 and ALT2 from ALT WWTP; SHZ1 and SHZ2 from SHZ WWTP; CJ1 and CJ2 from CJ WWTP; HX1 and HX2 from HX WWTP. Sampling date, flow rate, influents, effluents, and operational parameters of the WWTPs are presented in Table 1 . All WWTPs treat domestic wastewater, except for the SHZ WWTP. The influent of SHZ WWTP is composed of domestic and industrial wastewater, of which industrial wastewater accounts for 24%. SVI greater than 150 mL·g−1 is considered as bulking sludge [19 ]. Since the SVI of the samples from CJ and HX WWTPs were greater than 150 mL·g−1, the samples were bulking sludge, while the samples from ALT and SHZ WWTPs were normal sludge since their SVI were less than 150 mL·g−1 (Table 1 ). Additionally, the microscopic investigation showed that the sludge samples were filamentous bulking sludge.
The samples for the microbial analysis were stored in the laboratory at −40°C and were sent to Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) for DNA extraction, PCR amplification, and Illumina high-throughput sequencing.
The samples for the microbial analysis were stored in the laboratory at −40°C and were sent to Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) for DNA extraction, PCR amplification, and Illumina high-throughput sequencing.
7
Small and Total RNA Sequencing
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cDNA library preparation was conducted using the NEB small RNA library prep kit for small RNA sequencing and the NuGen Ovation RNAseq V2 kit, followed by the Ovation Ultralow V2 Library or Ovation Rapid Library Systems, for total RNA sequencing. Illumina high-throughput sequencing (HiSeq2500 SR 1 × 50 run and HiSeq3000 1 × 50) was applied to the samples (total number of reads for small RNA sequencing: 344508344; total number of reads for total RNA sequencing: 527413532) at the UCLA Microarray Core Facility.
8
RNA-Seq Analysis of FSTL3 Knockdown in HCT116 Cells
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The total RNA extracted from HCT116 cells was transfected with shFSTL3 and the control cells were lysed with TRIzol reagent. The cDNA library was constructed after the RNA samples were qualified. Immediately after the library was examined and passed, Illumina high-throughput sequencing was performed on the machine. The raw data of sequencing were evaluated for their quality and then compared with the reference genome for analysis. Finally, the gene expression data were collected. The above RNA-sequencing was performed by Aptbiotech Corp (Shanghai, China). The analysis of differentially expressed genes (DEGs) and Gene Ontology (GO) enrichment was carried out using R v3.6.1 (https://www.r-project.org/ ).
9
High-throughput sequencing of small RNAs
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The small RNA molecules (< 30 bases) were purified by PAGE and a pair of high-throughput sequencing adaptors were ligated to the 5′ and 3′ ends, then small RNA molecules were amplified for 17 cycles using adaptor primers. Fragments of 90 bp (small RNA+adaptors) were purified from an agarose gel. Purified DNA was used for cluster generation and sequencing analysis by Illumina high-throughput sequencing according to the manufacturer’s instructions. The data and results were generated as previously described [18 (link)]. Clean reads were compared using a miRBase database (release 20.0). The total copy number of each sample was normalized to 100,000.
10
High-throughput sequencing of CRISPR gRNA
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