Cellular suspensions were loaded on a 10X Chromium instrument. Gene expression libraries and feature barcoding libraries were prepared using Chromium Single Cell 3′ V3 Reagent Kits with Cell Surface Protein Feature Barcoding Technology for Cell Surface Protein (10X Genomics). VDJ libraries were prepared using Chromium Single Cell V(D)J V1 Reagent Kits (10X Genomics). The sequencing of gene expression libraries was conducted as a custom configuration (26x8x91bp) on the Illumina HiSeq3000 with a depth of 50,000 reads per targeted cell. The sequencing of VDJ libraries was conducted as PE150 on the Illumina HiSeq3000 with a depth of 5,000 reads per targeted cell. The sequencing of 10X feature barcode libraries was conducted as a custom configuration (26x8x25bp) on the Illumina HiSeq3000 with a depth of 5,000 reads per targeted cell. Junk cells with low read depth were removed and the remaining libraries were sequenced to a depth of 60M to 190M reads per sample in total.
Hiseq 3000
Manufactured by Illumina
Sourced in United States, China, France, Germany, Switzerland, Belgium, United Kingdom, Finland
The HiSeq 3000 is a high-throughput sequencing system designed for large-scale genomic projects. It offers a combination of speed, throughput, and accuracy to enable researchers to generate large amounts of sequencing data efficiently. The HiSeq 3000 is capable of sequencing multiple samples simultaneously, making it well-suited for applications such as whole-genome sequencing, targeted gene sequencing, and transcriptome analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (45 mentions)
Trizol reagent, by Thermo Fisher Scientific (44 mentions)
2100 bioanalyzer, by Agilent Technologies (41 mentions)
Rneasy mini kit, by Qiagen (40 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (38 mentions)
Hiseq 2500, by Illumina (27 mentions)
Novaseq 6000, by Illumina (23 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (22 mentions)
Bioanalyzer, by Agilent Technologies (21 mentions)
Truseq stranded mrna kit, by Illumina (18 mentions)
Rneasy micro kit, by Qiagen (18 mentions)
Nanodrop, by Thermo Fisher Scientific (18 mentions)
Bcl2fastq2 v 2.17 program, by Illumina (15 mentions)
Truseq stranded mrna sample prep kit, by Illumina (15 mentions)
Rneasy kit, by Qiagen (15 mentions)
Kapa library quantification kit, by Roche (15 mentions)
Qubit 2, by Thermo Fisher Scientific (14 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (12 mentions)
Nextera xt dna library preparation kit, by Illumina (12 mentions)
Ribominus eukaryote kit, by Qiagen (11 mentions)
1 014 protocols using hiseq 3000
1
Single-Cell Multimodal Sequencing Protocol
2
Comparative Transcriptomic Analysis of GBM and HCC
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The datasets for both GBM and HCC were taken from Gene Expression Omnibus (GEO). For GBM (normal samples-PRJNA494560 transcriptomic data with paired-end sequencing performed on Illumina HiSeq 3000 (Homo Sapiens) platform and tumor samples-PRJNA347513, transcriptomic data with paired-end sequencing performed on Illumina HiSeq 2000 platform) and for HCC (normal samples-PRJNA494560 transcriptomic data with paired-end sequencing performed on Illumina HiSeq 3000 (Homo Sapiens) platform and tumor samples-PRJNA414787 transcriptomic data with paired-end sequencing performed on Illumina HiSeq 2000 (Homo Sapiens) platform) were taken. The method that was followed for carrying out this study is shown below in flowchart (see Figure 1 ).
3
NGS Library Construction and Sequencing
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The NGS library was constructed using the SeqCap EZ System from NimbleGen (Arrowhead Madison, Inc. Madison, WN, USA) according to the manufacturer’s instructions. Briefly, genomic DNA was sheared to size the target sequences to roughly 300 base pairs that were converted to double-stranded DNA. The cDNA was then end repaired and specific oligonucleotide adapter ligated for multiplexing index and ligation-mediated polymerase chain reaction (LM-PCR), followed by incubation with SeqCap biotinylated DNA baits, and purification of the hybrids using streptavidin-coated magnetic beads. After amplification with a maximum of 18 cycles, the libraries were sequenced using 100-bp pair-ended reads on the Illumina HiSeq 3000 platform (Illumina, Inc., San Diego, CA, USA).
Libraries for RNA-Seq were prepared using a KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation, and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on an Illumina HiSeq 3000 for a single read 50 runs. Data quality check was done on an Illumina SAV. De-multiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program.
Libraries for RNA-Seq were prepared using a KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation, and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on an Illumina HiSeq 3000 for a single read 50 runs. Data quality check was done on an Illumina SAV. De-multiplexing was performed with the Illumina Bcl2fastq2 v 2.17 program.
4
RNA-Seq Library Preparation and Sequencing
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RNA-Seq was performed at the GeT-PlaGe core facility, INRA Toulouse, France. RNA-seq libraries were prepared according to Illumina’s protocols using the Illumina TruSeq Stranded mRNA sample prep kit. Briefly, after mRNA enrichment, 200 ng of mRNA were fragmented to generate double stranded cDNA and adapters were ligated. A total of 10 cycles of PCR were applied to amplify libraries. Library quality was assessed using an Advanced Analytical Fragment Analyzer and libraries were quantified by qPCR using the Kapa Library Quantification kit. RNA-seq experiments were performed on an Illumina HiSeq3000 using a paired-end read length of 2x150 bp with the Illumina HiSeq3000 chemistry. A total of 11 to 42 million paired-reads per sample was obtained, except for the sample EDL933_21 (122 million paired-reads) (Additional file 1 : Table S2).
5
PTBP1 Knockout Transcriptome and Binding
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To seek out differentially expressed genes upon PTBP1 knockout, total RNA was isolated from PTBP1 knockout or control MKN28 cells using Trizol reagent, and PolyA RNA was subsequently puri ed from total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq was performed to detect the mRNA expression pro les at GENTED (Shanghai, China) using HiSeq3000 (Illumina, USA). The differential genes were selected with fold change > 2 and a P-value < 0.05. To reveal PTBP1-bound mRNAs, RIP experiments were conducted using a PTBP1 antibody (Cell Signaling Technology) or IgG. Total RNA was isolated with Trizol (Invitrogen), and ribosomal RNA was removed from total RNA. RNAseq was performed at CLOUDSEQ (Shanghai, China) using HiSeq3000 (Illumina, USA). Data are available via GEO under accession numbers GSE157582 and GSE157941.
6
Targeted Enrichment and NGS Screening
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The patient sample was screened for disease-causing variants by a custom-designed targeted enrichment approach (HaloPlex™/Agilent Technologies) followed by next-generation sequencing on a HiSeq3000 (Illumina) platform as described previously [17 (link)]. In brief, enrichment of the targeted plus 25-bp flanking region was accomplished using the HaloPlex Target Enrichment System (Agilent Technologies Inc., 2013), based on a molecular inversion probe strategy. Library preparation was performed according to the manufacturer’s instruction. In brief, 200 ng of gDNA was digested by eight pairs of restriction enzymes, followed by bar code indexing and hybridization to custom-designed capture probes for 16 h at 54 °C. Thereafter, the circularized biotinylated target-probe complexes were extracted using magnetic streptavidin beads. The final steps included nick ligation, PCR library amplification, and AmPure XP bead (Beckman Coulter, Inc.) purification prior to qualitative and quantitative assessment of the DNA library using a 2100 Bioanalyzer instrument (Agilent). Next-generation sequencing was performed in a 150-bp paired-end mode using a HiSeq3000 (Illumina) platform.
7
PTBP1 Knockout Transcriptome Profiling
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To identify the differentially expressed genes upon PTBP1 knockout, the total RNA was isolated from the PTBP1 knockout or control MKN28 cells using TRIzol reagent, and PolyA RNA was subsequently purified from the total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module. RNA-seq was performed to detect the mRNA expression profiles at GENTED (Shanghai, China) using HiSeq3000 (Illumina, USA). The differentially expressed genes with a fold change > 2 and a P-value < 0.05 were selected. To reveal the PTBP1-bound mRNAs, RIP experiments were conducted using a PTBP1 antibody (Cell Signaling Technology) or IgG. The total RNA was isolated with TRIzol (Invitrogen), and ribosomal RNA was removed from the total RNA. RNA-seq was performed at CLOUDSEQ (Shanghai, China) using HiSeq3000 (Illumina, USA). The data are available via GEO under accession numbers GSE157582 and GSE157941.
8
Extracting and Sequencing RNA from Plant and Fungal Tissues
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Tree tissue samples were ground in liquid nitrogen using a bead beater and Lysing Matrix A (MP Biomedicals LLC, Solon, OH). Approximately 50 mg of each ground sample was used for RNA extraction using the method of Chang et al. [115 ]. The RNA was further treated with DNAase and concentrated using RNA Clean and Concentrator-5 (Zymo Research). RNA samples were submitted to GENEWIZ for library preparation and RNA sequencing (Illumina HiSeq3000, 2 × 150 bp, with PolyA Selection). For in vitro-grown fungal tissue, approximately 10 mg of the freeze-dried fungal tissue for each sample was homogenized using a bead beater. Total RNA was extracted using the RNeasy Plant Mini kit according to the manufacturer’s instruction (QIAGEN). The Interdisciplinary Center for Biotechnology Research (ICBR) NextGen DNA Sequencing core, University of Florida (UF) performed mRNA isolation using NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490) and RNA library construction with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, catalog # E7530) according to the manufacturer’s user guide. Paired-end, 2 × 100 cycle sequencing was performed at the ICBR on two lanes of the Illumina HiSeq3000 instrument using the clustering and sequencing reagents provided by Illumina (San Diego, CA, USA).
9
Bulk RNA Sequencing Workflow
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Total RNA from these samples were isolated using QIAzol Lysis Reagent (Qiagen, Valencia CA) and purified using miRNeasy MinElute Cleanup columns. RNA integrity was evaluated with per-sample RNA Integrity Number (RIN) using Agilent's Bioanalyzer 2100 and RNA 6000 Nano Kits.
Large RNA library preparation and sequencing was conducted at the Oklahoma Medical Research Foundation Genomics Core using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold (rRNAdepleted) preparation kits. The sequencing was conducted in ten separate batches (sequencing runs conducted on different flow cell and/or day), four in phase 1 and six in phase 2. In order to eliminate lane effects within each batch, samples were barcoded to distinguish samples and multiplexed across lanes. Sequencing of 2x150 paired-end reads was done using the Illumina Hiseq 3000 with target read depth of 40 million read pairs. Small RNA library preparation and sequencing at the University of California, Los Angeles, Clinical Microarray Core using New England Biolabs NEBNext library preparation, and sequencing of 1x50 single-end reads done on the Illumina Hiseq 3000 with target read depth of 15 million. Sequencing was conducted in twenty-seven batches, eleven in phase 1 and sixteen in phase 2, and samples were multiplexed within each batch to eliminate any lane artifacts.
Large RNA library preparation and sequencing was conducted at the Oklahoma Medical Research Foundation Genomics Core using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold (rRNAdepleted) preparation kits. The sequencing was conducted in ten separate batches (sequencing runs conducted on different flow cell and/or day), four in phase 1 and six in phase 2. In order to eliminate lane effects within each batch, samples were barcoded to distinguish samples and multiplexed across lanes. Sequencing of 2x150 paired-end reads was done using the Illumina Hiseq 3000 with target read depth of 40 million read pairs. Small RNA library preparation and sequencing at the University of California, Los Angeles, Clinical Microarray Core using New England Biolabs NEBNext library preparation, and sequencing of 1x50 single-end reads done on the Illumina Hiseq 3000 with target read depth of 15 million. Sequencing was conducted in twenty-seven batches, eleven in phase 1 and sixteen in phase 2, and samples were multiplexed within each batch to eliminate any lane artifacts.
10
Comparative Library Preparation of Fungarium DNA
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VPRI specimen 18536 was used as a DNA representative from each of the 13 DNA extraction protocols, in a comparison study of two library preparation kits, Illumina Nextera XT® (New England Biolabs) and NuGen Ovation® Ultralow System V2 (NuGen).
Illumina Nextera XT® double indexed and NuGen Ovation® single indexed sequencing library preparations were completed for 13 VPRI 18536 DNA samples as per manufacturer’s instructions (S1 File ). No DNA repair was performed on the fungarium DNA samples. The NuGen Ovation® Ultralow System V2 libraries DNA samples were fragmented to 350 bp by sonication using Covaris S-Series Focused ultrasonicator. Fragmentation sonication settings are shown in S2 File . DNA library concentrations were quantified using Promega Quantus™ fluorometer and Agilent 2200 TapeStation®. The finalised Illumina Nextera XT® and NuGen Ovation® Ultralow System V2 libraries were paired-end sequenced on the Illumina® HiSeq 3000 platform. Except for DneP+ Illumina Nextera XT® and NuGen Ovation® Ultralow System V2 libraries which were sequenced on Illumina® MiSeq using the reagent V3 600 cycles kit due to a changeover in sequencing platforms in our facility and Illumina® HiSeq 3000 is no longer available.
Illumina Nextera XT® double indexed and NuGen Ovation® single indexed sequencing library preparations were completed for 13 VPRI 18536 DNA samples as per manufacturer’s instructions (
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