10 HCT116-derived RNA samples were normalized and 200 ng of input was used for library construction using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760), together with the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) upstream and the NEXNext Multiplex Oligos for Illumina (E7600) downstream (all New England Biolabs). Sequencing libraries were quantified, normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot; and sequenced on a HiSeq 4000 Sequencing System (Illumina) with dual index, single read at 50 bp length (Read parameters: Rd1: 51 Rd2: 8, Rd3: 8), reaching an average depth of 40 million Pass-Filter reads per sample (11.1% CV).
Hiseq 3000 4000 sr cluster kit
Manufactured by Illumina
The HiSeq 3000/4000 SR Cluster Kit is a laboratory equipment product designed for the Illumina HiSeq 3000 and HiSeq 4000 sequencing platforms. The kit is used to prepare samples for high-throughput sequencing. It provides the necessary reagents and materials to generate clusters, which are essential for the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
Truseq stranded mrna library prep kit, by Illumina (3 mentions)
Nebnext poly a mrna magnetic isolation module, by Illumina (2 mentions)
Phix control v3, by Illumina (2 mentions)
Hiseq 3000 4000 sbs kit, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000 sequencing system, by Illumina (1 mentions)
Dnase 1, by New England Biolabs (1 mentions)
Monarch total rna miniprep kit, by New England Biolabs (1 mentions)
Truseq stranded mrna sample preparation guide, by Illumina (1 mentions)
Hiseq 3000 platform, by Illumina (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by Illumina (1 mentions)
High sensitivity dsdna quanti it assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 3000 sequencing system, by Illumina (1 mentions)
12 channel fragment analyzer, by Agilent Technologies (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
5 protocols using hiseq 3000 4000 sr cluster kit
1
HCT116 RNA-seq Library Prep Protocol
2
Transcriptome Profiling of Leaf Tissue
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Total RNA was extracted from ground leaf tissue using the Monarch® Total RNA Miniprep Kit (New England Biolabs Inc.) following the manufacturer’s instructions. Contaminating DNA was digested with DNase I (New England Biolabs Inc.). The integrity of input RNA was analysed on a 2100 Bioanalyzer (Agilent). Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina) following the Illumina TruSeq Stranded mRNA Sample Preparation Guide #15031047 Rev. E, with the following adaptations: 300 ng total RNA as the basis for library preparation, adapter index dilution to 50% with RNase-free water, and an additional purification step with AMPure XP (Beckman Coulter).
The concentration of the resulting libraries was estimated using a NanoDrop 8000 (Thermo Fisher Scientific) and quality control was performed with a High Sensitivity NGS Fragment Analysis Kit (1 bp–6000 bp) DNF-474 (Advanced Analytical Technologies, Inc.). Based on quantification with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), libraries were adjusted to 2 nM concentration before cluster generation with a HiSeq 3000/4000 SR Cluster Kit (Illumina). Twelve samples were pooled per lane and sequenced in 150 bp single-end mode on a HiSeq 3000 platform (Illumina). On average, over 32 million reads per library were obtained (Supplementary Table S1 ).
The concentration of the resulting libraries was estimated using a NanoDrop 8000 (Thermo Fisher Scientific) and quality control was performed with a High Sensitivity NGS Fragment Analysis Kit (1 bp–6000 bp) DNF-474 (Advanced Analytical Technologies, Inc.). Based on quantification with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), libraries were adjusted to 2 nM concentration before cluster generation with a HiSeq 3000/4000 SR Cluster Kit (Illumina). Twelve samples were pooled per lane and sequenced in 150 bp single-end mode on a HiSeq 3000 platform (Illumina). On average, over 32 million reads per library were obtained (
3
RNA-seq Analysis of Macrophage Enhancers
4
RNA-Seq Library Preparation and Sequencing
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MDSC-derived RNA samples were normalized, and a RNA input of 100 ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEXNext Multiplex Oligos for Illumina #E7600 downstream (New England Biolabs, Frankfurt am Main, Germany). Ampure XP beads (Beckman Coulter, Brea, CA, USA) were used for double-stranded cDNA purification. mRNA sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher Scientific) on a Synergy HTX (BioTek). Library molarity averaged at 134 nM. Final library size distribution was assessed (smear analysis of 364 bp average and adapter dimer presence <0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 12-channel Fragment Analyzer (Agilent Technologies). All sequencing libraries passed quality check, were normalized, pooled, and spiked in with PhiX Control v3 (Illumina, San Diego, CA, USA). The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot and sequenced on a HiSeq 3000 Sequencing System (Illumina) with single index, single read at 85 bp length (Read parameters: Rd1: 85, Rd2: 8), reaching an average depth of 29 million Pass-Filter reads per sample (11% CV).
5
RNA-seq Analysis of Macrophage Enhancers
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RNA-seq sequencing libraries were made from 1 µg of DNase-treated total RNA samples using TruSeq stranded mRNA Library Prep Kit (Illumina, RS-122-2101, RS-122-2102) and performed according to the manufacturer’s instructions. RNA-seq libraries were quantified with Qubit, clustered and sequenced using HiSeq 3000/4000 SR cluster kit (Illumina GD-410-1001) and HiSeq 3000/4000 SBS kit (Illumina FC-410-1001). RNA-seq reads were counted by HOMER software considering only exonic regions for RefSeq genes. The proximal macrophage-expressed genes of 6,775 macrophage-restricted enhancers were obtained by using these criteria: FC≥4, over 20 tags in macrophage, and visualized by heatmap with log2 transform. Gene ontology analysis and genetic association analysis was performed using Metascape (http://metascape.org ).
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