Illumina offers a wide range of powerful library preparation kits that provide fast and easy library preparation workflow, and the Illumina HiSeqTM 4000 sequencing results are highly technically repeatable. However, to further verify the accuracy of our peripheral white blood cell sample sequencing results, we used qRT-PCR to validate 5 hub genes (CXCL8, EGR1, EGR3, IL1B, and PTGS2) in the Turquoise module to evaluate whether the results were consistent with mRNA sequencing results and to verify the accuracy of the Illumina HiSeqTM 4000 sequencing results. Extracted RNA was synthesized with complementary DNA (cDNA) using a reverse transcription kit (Takara). Real-time PCR was carried out with TB Green series kit (Takara) and monitored with the Bio-RAD (CFX96 real-time) system. β-actin for mRNA was applied to normalize the result. All reactions were repeated 3 times and relative gene expressions were evaluated by the 2−ΔΔCt method. Primer sequences are shown in Table 1 .
Hiseq 4000 sequencing
Manufactured by Illumina
Sourced in China, United States
The HiSeq 4000 is a high-throughput DNA sequencing system produced by Illumina. It is designed to generate large amounts of sequencing data efficiently. The HiSeq 4000 uses Illumina's proprietary sequencing-by-synthesis technology to sequence DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (7 mentions)
Bz x710 microscope, by Keyence (2 mentions)
Epicult b medium, by STEMCELL (2 mentions)
Fluidigm c1 suspension reagents, by Standard BioTools (2 mentions)
Nanophotometer spectrophotometer, by Agilent Technologies (2 mentions)
Total rna kit, by Tiangen Biotech (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by Illumina (1 mentions)
Truseq rna sample preparation kit, by Illumina (1 mentions)
Paxgene blood rna tube, by BD (1 mentions)
Sigma dna extraction kit, by Merck Group (1 mentions)
Truseq stranded mrna sample preparation kit, by Illumina (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Low range agarose, by Bio-Rad (1 mentions)
Hiseq 2500 sequencing platform, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
36 protocols using hiseq 4000 sequencing
1
Validating Illumina HiSeq Sequencing Data
2
RNA-seq Library Preparation and Sequencing
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RNA-seq library preparation utilized the TruSeqTM RNA sample preparation kit (Illumina, USA). Then, rRNA-free RNA samples were incubated with fragmentation buffer, and the SuperScript double-stranded cDNA synthesis kit (Invitrogen) was employed for cDNA synthesis. Amplification utilized Phusion DNA polymerase (New England Biolabs), and Illumina HiSeq4000 sequencing was then performed by Majorbio Biotech (China) (58 (link)). The PE150 sequencing strategy was selected, with a 250-300 bp insert strand specific library.
3
RNA-seq Analysis of Whole Blood
4
Single-cell RNA-seq of Mammary Cell Populations
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For scRNA-seq using the 10X Chromium system, FACS-sorted mammary basal and luminal cell populations were combined. Library generation and Illumina HiSeq-4000 sequencing were performed as previously described (Haensel et al., 2020 (link)). For scRNA-seq using the Fluidigm C1 platform, FACS-sorted basal MECs (Lin−CD49fhighEpCAM+) were washed and resuspended with Epicult-B medium (Stem Cell Technologies; Ca. No. 05610) at a concentration of ~500 cells/μL. Cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100–5315) at a ratio of 8:2 before loading onto the C1 chip (Fluidigm 100–5760). Bright-field images of captured cells were collected using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol #100–7168 I1. RNA spike-in controls were omitted. cDNA library preparation was performed following the Fluidigm C1 Protocol #100–7168 I1.
Data analysis was performed using Seurat as we previously described (Haensel et al., 2020 (link)). Heatmaps were based on normalized, but not raw, values of expression to enable direct comparison across runs. A color gradient depicting the expression of each gene in each cell per average for all the cells was generated for each analysis combining biological replicates.
Data analysis was performed using Seurat as we previously described (Haensel et al., 2020 (link)). Heatmaps were based on normalized, but not raw, values of expression to enable direct comparison across runs. A color gradient depicting the expression of each gene in each cell per average for all the cells was generated for each analysis combining biological replicates.
5
Genomic DNA Extraction and Sequencing
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Total DNA of each variety was extracted from leaves of two-week-old germinated seedling using Mutou et al.'s protocol [42 (link)] and Sigma DNA extraction kit. DNA quality and quantity were analysed using NanoDrop spectrophotometer. The integrity of DNA samples was determined using 0.8% agarose gel. The DNA samples were sequenced using Illumina HiSeq 4000 sequencing (Illumina, Inc., San Diego, CA, USA). Standard Illumina protocol was used for the sequencing process.
6
RNA-seq Analysis of Sarcoma Samples
7
Illumina TruSeq Stranded mRNA Library Prep
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Libraries for RNA‐seq were prepared by following the manufacturer's protocol for the Illumina TruSeq Stranded mRNA Sample Preparation Kit (RS‐122‐2103, Illumina). In brief, 3 μg of total RNA was used for library construction. Poly(A) RNA was captured by oligo‐dT beads and fragmented upon elution from the beads. The first‐strand cDNA was synthesized by SuperScript III reverse transcriptase (Invitrogen) using dNTPs and random primers. The second‐strand cDNA was generated using a dUTP mix. A single A base was added to the 3′ end of the double‐stranded (ds) cDNA, before ligating barcoded Truseq adapters. The resulting products were purified and enriched by means of 12 cycles of PCR to create the final double‐stranded cDNA library. A final size selection was performed by means of 1.4% low‐range agarose (Bio‐Rad) gel electrophoresis to yield a library of inserts 200–400 bases in length. The library was extracted from the agarose gel using a MinElute PCR Purification Kit (Qiagen). Final libraries were analysed using a Fragment Analyser HS NGS Fragment Kit (Agilent) to estimate quantity and check size distribution. The prepared libraries were pooled and subjected to Illumina HiSeq 4000 sequencing.
8
High-throughput Sequencing of Duck Transcriptome
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Genomic DNA was extracted using the standard phenol/chloroform extraction method. For
each sample, two paired-end libraries (500 bp) were constructed according to the
manufacturer's protocols (Illumina) and sequenced on the Illumina Hiseq 2500 sequencing
platform. We sequenced each sample at 5X depth in order to reduce the false-negative rate
of variants due to our strict filter criteria. We randomly selected one individual for 10X
coverage, except for the MDN population, where we sequenced seven individuals at 5X
coverage and random one at 20X coverage and the MDZ population, where we sequenced all
individuals at 10X coverage. We generated 628.37 Gb of paired-end reads of 100 bp (or 150
bp; MDZ) length (Supplemental Table
S1 ).
The mRNA from brain, liver, and breast muscle of 14 ducks were extracted using the
standard trizol extraction methods. For each sample, two paired-end libraries (500 bp)
were constructed according to the manufacturer's instruction (Illumina). All samples were
sequenced using Illumina Hiseq 4000 sequencing platform with the coverage of 6X. We
generated 278.62 Gb of paired-end reads of 150 bp length (Supplemental Table S9 ).
each sample, two paired-end libraries (500 bp) were constructed according to the
manufacturer's protocols (Illumina) and sequenced on the Illumina Hiseq 2500 sequencing
platform. We sequenced each sample at 5X depth in order to reduce the false-negative rate
of variants due to our strict filter criteria. We randomly selected one individual for 10X
coverage, except for the MDN population, where we sequenced seven individuals at 5X
coverage and random one at 20X coverage and the MDZ population, where we sequenced all
individuals at 10X coverage. We generated 628.37 Gb of paired-end reads of 100 bp (or 150
bp; MDZ) length (
S1
The mRNA from brain, liver, and breast muscle of 14 ducks were extracted using the
standard trizol extraction methods. For each sample, two paired-end libraries (500 bp)
were constructed according to the manufacturer's instruction (Illumina). All samples were
sequenced using Illumina Hiseq 4000 sequencing platform with the coverage of 6X. We
generated 278.62 Gb of paired-end reads of 150 bp length (
9
Amplifying and Sequencing CHIKV RNA from Mosquitoes
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Amplicon libraries were prepared as previously described [45 (link)]. Briefly, CHIKV RNA isolated from bodies and saliva was amplified by nine separate RT-PCR reactions using a high-fidelity RT-PCR kit and the primers described in Riemersma et al., 2019 [45 (link)]. The nine overlapping cDNA amplicons spanning from the 5’ to 3’ untranslated regions (UTR), excluding the first 14 nucleotides of the 5’ UTR and the last 55 nucleotides of the 3’ UTR, were pooled at equimolar ratios and enzymatically fragmented to approximately 150 bp. Illumina sequencing libraries were prepared with the fragmented cDNA using a NEBNext Ultra II DNA library prep kit and NEBNext Multiplex Oligos (New England Biolabs). Libraries of P4 infectious plasmid DNA with (pY_PCR) and without (pY) RT-PCR amplification, and P4 in vitro transcribed RNA were used as sequencing controls. An unrelated library (WNV cDNA) was also included as a control for index hopping with Illumina HiSeq 4000 sequencing [56 ]. All libraries were sequenced in parallel with paired-end 150 reads on a single flow cell lane of an Illumina HiSeq 4000 instrument at the UC Davis DNA Technologies Core. Raw fastq files are available from NCBI Sequence Read Archive under BioProject entry PRJNA541092.
10
Single-cell RNA-seq of Mammary Cell Populations
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