We used the samples harvested after one and three days for transcriptome sequencing. The TRIzol method was used for total RNA extraction. The frozen muscle tissue was immersed with TRIzol™ Reagent (Thermo-Fisher Scientific, Waltham, MA, USA) and was crushed using a tissue crusher until there were no visible solids to ensure that the muscle tissue was completely dissolved. The RNA was extracted with chloroform and isopropanol in sequence, eluted with 75% ethanol, and finally dissolved in diethylpyrocarbonate water. We used 2 μg total RNA to enrich mature mRNA using magnetic beads carrying Oligo (dT), and the enriched RNA was interrupted after purification. Random primer fragments were used as a template to synthesize the first-strand cDNA, which was then used as a template to synthesize double-stranded DNA. A series of treatments such as end repair, A-tailing, and connection of sequencing adapters were performed, and the samples were amplified using polymerase chain reaction to complete library construction. Quality testing was conducted using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The Illumina HiSeq X Ten sequencer (Illumina Inc., San Diego, CA, USA) was used for sequencing, and 150 bp paired-end data was obtained. The gene expression dataset is available on ArrayExpress and the accession number is E-MTAB-10794.
Hiseq x ten sequencer
Manufactured by Illumina
Sourced in United States, China
The HiSeq X Ten sequencer is a high-throughput DNA sequencing system designed and manufactured by Illumina. It is capable of generating large amounts of genomic data by simultaneously processing multiple samples. The core function of the HiSeq X Ten is to perform DNA sequencing, which is the process of determining the precise order of nucleotides within a DNA molecule.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (16 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (16 mentions)
Dneasy tissue kit, by Qiagen (7 mentions)
Truseq rna sample preparation kit, by Illumina (6 mentions)
End repair mix, by Thermo Fisher Scientific (6 mentions)
2100 bioanalyzer, by Agilent Technologies (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Dneasy blood tissue kit, by Qiagen (5 mentions)
Trizol, by Thermo Fisher Scientific (5 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (5 mentions)
Truseq rna sample prep kit, by Illumina (4 mentions)
Dnase 1, by Takara Bio (4 mentions)
Ampure xp bead, by Beckman Coulter (4 mentions)
Nebnext ultra rna library prep kit for, by Illumina (3 mentions)
Picogreen dsdna assay, by Thermo Fisher Scientific (3 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
Kapa hyper prep kit, by Roche (3 mentions)
Truseqtm rna sample preparation kit, by Illumina (3 mentions)
Nebnext ultra rna library prep kit, by New England Biolabs (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
183 protocols using hiseq x ten sequencer
1
Transcriptome Profiling of Muscle Tissue
2
RNA-Seq Analysis of Lhx4 Null Retinas
3
Genomic DNA Extraction and Whole-Genome Sequencing
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Whole-blood-derived genomic DNAs were extracted from all individuals using the QIAamp DNA blood Midi Kit (QIAGEN GmbH, Hilden, Germany) with standard protocols. The DNA purity was detected using NanoPhotometer spectrophotometer (IMPLEN, CA, USA). The DNA quantity was assessed using the Qubit Fluorometer (Life Technologies, CA, USA). Next, 1 ug of high quantity genomic DNA was fragmented. The fragments were then end repaired, poly-A tailed and adapter ligated using TruSeq DNA Sample Preparation Kit (Illumina, 15026486 Rev.C), according to the manufacturer’s instructions. Adapter-ligated libraries were amplified by 6 cycles of PCR. After that, libraries were assessed for sequencing using Agilent 2100 Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA, USA). Next, the qualified libraries were sequenced using Illumina HiSeq X Ten sequencer from Illumina (Illumina, San Diego, CA, USA) with 2 × 150 bp paired-end reads.
4
RNA Extraction and Illumina RNA-seq Library Preparation
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RNA was extracted using a plant RNA Isolation kit (DP432, Tiangen Technologies, Beijing, China) following manufacturer’s instructions. RNA quality and purity were assessed using Agilent 2100 (Agilent Technologies, CA, USA) and NanoDrop One (Thermo Fisher, DE, USA). RNA with the Ratios of OD260/OD280 above 2.0 was used in the next experiment. Illumina RNA-seq libraries were created from 10 μg RNA from each sample using the NEBNext UltraTM RNA Library Prep Kit (New England BioLabs, MA, USA) according to manufacturer's instructions. Resulting libraries were further assessed using the Agilent 2100 (Agilent Technologies, CA, USA). The qualified libraries were then sequenced on an Illumina HiSeq X-ten sequencer (Illumina, CA, USA) at BioMarker Technologies (Beijing, China).
5
RNA-Seq Analysis of Lhx4 Null Retinas
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For RNA-Seq experiment at each developmental stage (P6 and P7), three Lhx4 null mice and three control mice were used, and the retinas of each mouse were collected as one RNA-Seq sample. Retinas were isolated by removing retinal pigment epithelium from eyecups under a dissecting microscope. Total RNA was extracted with RNeasy kits (QIAGEN, Valencia, CA) in accordance with the manufacturer’s protocol. The cDNA libraries were generated by using TruSeq RNA Sample Prep Kit (Illumina) and were sequenced on the Illumina HiSeq X ten sequencer (Illumina). Sequence depths are from 19M to 40M reads per library and sequence parameters were set at 150 bp paired-end reads. Short reads mapping and differentially expressed gene identification were performed using Tophat (RRID: SCR_013035) and Cufflinks (RRID: SCR_014597) (Trapnell et al., 2012 (link)). Gene ontology enrichment was analyzed by using Enrichr (http://amp.pharm.mssm.edu/Enrichr/ ; Kuleshov et al., 2016 (link)). The accession number for the RNA-seq data from Lhx4 null retinas and control reported in this paper is GEO: GSE126942 and GSE127771.
6
Transcriptome Analysis of Cowpea Transgenics
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Four RNA sequencing libraries (CL, DL, CR, DR) were prepared with Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs, MA, USA) according to manufacturer's instructions (Genotypic Technology Pvt. Ltd., Bangalore, India). Similarly, six RNA sequencing libraries prepared from leaf and root of control and T 2 generation transgenic amiR408-OE lines 1 and 5 were analysed using Illumina HiSeq platform (Clevergene Biocorp Private Limited, Bangalore, India). Brie y, mRNA isolation was performed from 1 µg total RNA using oligo-dT magnetic beads and further, subjected to fragmentation and priming followed by cDNA synthesis. The cDNA fragments were ampli ed to generate transcriptome libraries and sequenced on Illumina HiSeq XTen sequencer (Illumina, San Diego, USA) for 150 bp paired-end chemistry following manufacturer's procedure. The sequencing quality was assessed using FastQC v0.11.8 software. Transcriptome analysis was performed by processing the raw data for removal of low-quality data (
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7
Single-Cell RNA-Seq with 10x Genomics
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Single cell barcoding and library preparation were performed based on 10×Genomics single-cell RNA sequencing platform (10×Genomics, Pleasanton, CA, USA). Briefly, the single cell suspension prepared above were immediately counted using a hemocytometer (TC20, Bio-Rad, Hercules, CA, USA) and the cell concentrations were adjusted to 1,000 cells/μl prior to barcoding. To barcode the single cells with 10×Barcoded gel beads, 10×Genomics Chromium Single Cell 3' Library & Gel Bead Kit v2 (10×Genomics Inc., Pleasanton, CA, USA, 120237) and 10×Genomics Chromium barcoding system was used to construct 10×barcoded cDNA library following the manufacturer's instructions. Illumina HiSeq X Ten sequencer (Illumina, San Diego, CA, USA) was used for sequencing and pair-ended 150 bp (PE150) reads were generated for downstream analysis.
8
Variant Identification and Annotation Pipeline
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After sequencing, raw data were saved in FASTQ format. Illumina sequencing adapters and low quality reads (< 80 bp) were filtered by Cutadapt [14 ]. Clean reads were aligned to UCSC hg19 human reference genome using the Burrows-Wheeler Alignment [15 (link)] tool. Duplicated reads were removed using Picard (http://broadinstitute.github.io/picard ). Insertions, deletions and SNP variants were detected and filtered using the Genome Analysis Toolkit [16 (link)]. Then the identified variants were annotated using ANNOVAR [17 (link)] and associated with the following databases: 1000 genomes, Exome Aggregation Consortium, The Human Gene Mutation Database, and predicted by Mutation Taster (MT) [18 (link)], Sorting Intolerant From Tolerant (SIFT) [19 (link)], PolyPhen-2 (PP2) [20 (link)] and Genomic Evolutionary Rate Profiling (GERP++) [21 (link), 22 (link)]. Splice-site were predicted by Human Splicing Finder [23 (link)]. All variants identified by the Illumina HiSeq X Ten sequencer were confirmed by Sanger sequencing. The pathogenicity of mutations was assessed in accordance with American College of Medical Genetics and Genomics guideline (ACMG) [9 (link)–11 (link)].
9
Transcriptomic Analysis of Toxoplasma Tachyzoites
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Freshly egressed tachyzoites were collected and purified through polycarbonate membranes with a pore size of 3 μm. Samples were washed with PBS, and total RNA was extracted using the TRIzol methods according to the manufacturer’s instructions (TransGen Biotech, Beijing, China). Three samples were independently prepared for each strain. Then, RNA libraries were constructed using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA) and sequenced with the Illumina HiSeq X Ten sequencer.
The raw sequencing reads were trimmed and quality controlled by SeqPrep and Sickle. Then, clean reads were mapped to the reference genome ME49 (downloaded from ToxoDB) using TopHat. To identify differentially expressed genes, the transcript level for each gene was calculated according to the fragments per kilobase of exon per million mapped reads (FPKM) method and then compared.
To validate the gene expression changes revealed by RNA-Seq, RNA samples were also subjected to quantitative RT-PCR analysis using the Fast SYBR method (Vazyme Biotech Co., Nanjing, China). Primers used are listed in Table S1. Expression of target genes was normalized to that of β-tubulin and then compared. Each sample was independently analyzed three times, each in triplicate.
The raw sequencing reads were trimmed and quality controlled by SeqPrep and Sickle. Then, clean reads were mapped to the reference genome ME49 (downloaded from ToxoDB) using TopHat. To identify differentially expressed genes, the transcript level for each gene was calculated according to the fragments per kilobase of exon per million mapped reads (FPKM) method and then compared.
To validate the gene expression changes revealed by RNA-Seq, RNA samples were also subjected to quantitative RT-PCR analysis using the Fast SYBR method (Vazyme Biotech Co., Nanjing, China). Primers used are listed in Table S1. Expression of target genes was normalized to that of β-tubulin and then compared. Each sample was independently analyzed three times, each in triplicate.
10
Genome Sequencing of Ceracris nigricornis
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Total genomic DNA was extracted from the flight muscle and testes using a Genomic‐tip 20/G kit (QIAGEN), following the manufacturer's protocol. The quality of DNA samples was assessed with Qubit (Thermo Fischer Scientific), NanoPhotometer (Implen), and 2200 TapeStation (Agilent) instruments. For genome assembly, the genomic DNA of an SI individual (SI03) was sent to GeneBay, Inc. (Yokohama) for library construction and long‐read sequencing using the ONT PromethION system (Oxford Nanopore Technologies). Short‐read sequencing was performed with 150‐bp paired‐end reads in three runs on an Illumina HiSeq X Ten sequencer (Illumina) at Macrogen Japan. For genome assembly, a single sample (SI03) was sequenced using one lane of the HiSeq X Ten sequencer. For resequencing, 10 samples from the SI and ER groups and 15 samples from the WR, SG, and SR groups were separately sequenced using two lanes of the HiSeq X Ten sequencer. Library construction for SI and ER samples was performed at Macrogen Japan and that for WR, SG, and SR samples was performed at Kyoto University using the NEB Next Ultra II FS DNA Library Prep Kit for Illumina (E7805S; New England Biolabs). Adapters on raw reads were trimmed using Porechop (v0.2.4, https://github.com/rrwick/Porechop ) for ONT PromethION long reads and fastp (Chen et al., 2018 (link)) for Illumina HiSeq X short reads.
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