Genome sequencing was performed by Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). The genome sequence of the strain XT1-2-2 was obtained via the Illumina Hiseq×10 and Pacbio platforms, with a depth of ~ 100-fold coverage in both platforms. The previously extracted genomic DNA was randomly fragmented through Covaris or Bioruptor method. Fragmented DNA was purified by the QIAquick Nucleotide Removal Kit (Qiagen, Crawley, United Kingdom). Sequencing adaptors were ligated to A-tailed 3’ends according to the manufacturer’s instructions. A library for Illumina Paired-End sequencing was prepared. The sequencing library was sequenced via the combined sequencing method of Illumina Hiseq ×10 + PacBio, and each sample provides at least 100× PacBio sequencing data and 100× Illumina sequencing data of the genome to ensure a more complete and accurate assembly. The constructed standard shotgun library generated 165575 reads totaling 164774594 bp and an average length of 7034.7 bp. The resulting reads were de novo assembled with the help of SOAPdenovo v1.05 [37 (link)]. The genome was annotated using the NCBI Pro-karyotic Genome Annotation Pipeline (PGAP), and genes were identified by the gene caller GeneMarkS (Version 4.3). The genomic islands were predicted by Island Viewer 4 [38 (link)].
Hiseq 10
Manufactured by Illumina
Sourced in China, United States
The HiSeq ×10 is a high-throughput sequencing system designed for large-scale genomic analysis. It provides a flexible and scalable platform for a wide range of applications, including whole-genome sequencing, exome sequencing, transcriptome analysis, and more. The HiSeq ×10 system leverages Illumina's proven sequencing-by-synthesis technology to deliver high-quality, accurate data.
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Lab products found in correlation
Ga pipeline version 1.6, by Illumina (3 mentions)
Random hexamer primers, by Illumina (2 mentions)
Superscript double stranded cdna synthesis kit, by Illumina (2 mentions)
Truseqtm rna sample preparation kit, by Illumina (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (1 mentions)
Novaseq, by Illumina (1 mentions)
Truseq stranded total rna library prep kit, by Illumina (1 mentions)
Indices 1 12, by Illumina (1 mentions)
Nebnext ultra 2 directional rna seq kit, by New England Biolabs (1 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
Poly a rna selection kit, by Lexogen (1 mentions)
Sureselect human all exon v6 kit, by Agilent Technologies (1 mentions)
Nova sequencing platform, by Illumina (1 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
Bigdye terminator v3, by Thermo Fisher Scientific (1 mentions)
Soapdenovo2, by Illumina (1 mentions)
Hiseq x10, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Nebnext ultratm 2 rna library prep kit, by New England Biolabs (1 mentions)
23 protocols using hiseq 10
1
Genome Sequencing of Strain XT1-2-2
2
RNA-seq Analysis of Arabidopsis Seedling Mutants
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Total RNAs extracted from 15-day-old seedlings of wild type and mutants were sent to Novogene, China, for mRNA-seq library construction and the libraries were sequenced on an Illumina Hiseq ×10 platform to generate single-end reads of 150 bp in length. The analysis was performed by a homemade pipeline PIHSDA (https://github.com/grubbybio/PIHSDA/blob/master/pihsda ). Clean reads were trimmed to remove adapter sequences and mapped to Arabidopsis genome TAIR 10 using HISAT2 with default settings52 (link). Transcript levels were calculated and normalized in RPM. Then gene expression levels were normalized and differentially expressed genes (DEGs) were calculated with the cut-off of fold-change > 1.5 (for hyper DEGs) or fold-change < 0.67 (for hypo-DEGs), and P < 0.05 using DEseq253 (link). For GO-terms analysis, only genes with fold-change > 2.0 (for hyper DEGs) or fold-change < 0.5 (for hypo-DEGs), P < 0.01, and RPM > 10 in either genotype (average of three replicates for each genotype) were included. For heatmap figures, R genes with fold-change > 1.5 (for hyper DEGs) or fold-change < 0.67 (for hypo-DEGs), P < 0.05, and RPM > 10 in either genotype (average of three replicates for each genotype) were included. The GO-term enrichment analysis was performed online at TAIR (http://www.arabidopsis.org/tools/go_term_enrichment.jsp ). Only the top 20 terms were presented in this paper.
3
Illumina Sequencing and Variant Calling of Switchgrass
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The samples were sequenced by using Illumina HiSeq ×10 and Illumina NovaSeq 6000 paired-end sequencing (2 × 150 bp) at HudsonAlpha Institute for Biotechnology, Huntsville, AL, and the Department of Energy Joint Genome Institute, Berkeley, CA. To account for variable library sizes, reads were pruned to a max coverage of 50×. Reads were mapped to the P. virgatum v5 assembly (18 (link)) by using bwa-mem (64 (link)). Duplicate reads were filtered by using Picard (http://broadinstitute.github.io/picard) and realigned around indels by using GATK 3.0 (65 (link)). Multisample SNP calling was done by using SAMtools mpileup (66 (link)) and Varscan V2.4.0 (67 (link)) with a minimum coverage of eight and a minimum alternate allele count of four. Alleles were determined by using a binomial test (hypothesized probability of success = 0.5). However, for the tetrasomic calls, a binomial test was performed for three hypothesized probabilities (0.25, 0.5, and 0.75) using the binom.test function in R based on a maximum P value and the contribution of reads that match reference: Five tetrasomic genotypes were determined, equivalent to AAAA, AAAB, AABB, AAAB, and BBBB (or 0/4; 1/3; 2/2; 3/1; and 4/0).
4
SMART-seq2 for Single-cell RNA-seq
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Single-cell RNA sequencing was performed using the SMART-seq2 protocol [27 (link)] with minor modification. Briefly, a single cell was first lysed in 0.5 µl lysis buffer and RNAs were converted using Superscript III. After purification, 0.1 ng amplified cDNA was used for library construction. Libraries were then sequenced on Illumina HiSeq ×10 in paired-end, 150 bp mode.
5
Illumina Sequencing of PCR Products
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Polymerase chain reaction products were further purified by agarose gel electrophoresis according to the specification of the manufacturer, the purified PCR products were sequenced on the Illumina HiSeq × 10 platform (Illumina, San Diego, CA, USA) using specialized sequencing primers with various sample barcodes, and with a read length of 2 × 150 bps.
6
Single-cell RNA-seq of human immune cells
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The FACS-sorted viable hCD45+ cells were resuspended at 5 × 105–1 × 106 cells/ml with final viability of >85%. Single-cell library preparation was carried out using the 3′ library v2 or 5′ V(D)J and gene expression platform as per the 10× Genomics protocol (10× Genomics, Pleasanton, CA, USA). Cell suspensions were loaded onto a Chromium Single-Cell Chip along with the reverse transcription master mix and single-cell gel beads, aiming for 2000–10,000 cells per channel. Following the generation of single-cell gel bead-in-emulsions (GEMs), reverse transcription, and amplification were performed. Then, amplified cDNAs were purified and sheared. Sequencing libraries were generated with a unique sample index for each sample. Libraries were sequenced by Illumina HiSeq ×10 or NovaSeq.
7
KAPA Hyper Prep Kit Library Construction
8
Illumina TruSeq Stranded Total RNA Library Prep
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The TruSeq™ Stranded Total RNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to construct the library for the experiment. After removing rRNA and adding fragmentation buffer, mRNA was randomly broken into small fragments of approximately 200 nt. Under the action of reverse transcriptase, one-strand cDNA was synthesized using random primers and mRNA as templates. For the second strand synthesis, dUTP was used instead of dTTP to form the base of the second strand of cDNA containing dTTP. Before PCR amplification, the second strand of cDNA was digested with the UNG enzyme so that only the first strand of cDNA was included in the library. Finally, Illumina Hiseq × 10 (2 × 150 bp read length) was used for sequencing. Processing of the original images to sequences, base-calling, and quality value calculations were performed using the Illumina GA Pipeline (version 1.6), in which 150 bp paired-end reads were obtained.
9
Transcriptome Analysis of heso1 urt1 ski2 Mutants
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Total RNAs extracted from mature leaves of heso1 urt1 ski2 and other genotypes were sent to Berry Genomics (Beijing, China), for RNA-seq library construction. The libraries were sequenced on an Illumina Hiseq ×10 platform to generate pair-end reads of 150 bp in length. The RNA-seq data were analyzed using pRNASeqTools (https://github.com/grubbybio ). DEGs were calculated with the cutoff of fold change > 2 (for hyper-DEGs) or fold change < 0.5 (for hypo-DEGs), and P < 0.05. Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed using the R package clusterProfiler.
10
RNA-Seq Library Preparation Protocol
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Libraries were prepared from total RNA by using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, UK). Poly(A)-tailed mRNAs were isolated using a Poly(A) RNA Selection Kit (LEXOGEN, Austria). The isolated mRNAs were used for the synthesis of cDNA, which was then sheared, following the manufacturer’s instructions. Indexing was performed using the Illumina indices 1-12 (Illumina, USA). The enrichment step was carried out using polymerase chain reaction (PCR). Subsequently, libraries were checked using the Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) to evaluate the mean fragment size. Quantification was performed using the library quantification kit using a StepOne Real-Time PCR System (Life Technologies, USA). High-throughput sequencing (paired-end 100 bp) was performed using HiSeq ×10 (Illumina).
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