Hiseq 3000 4000 sbs kit reagents

Manufactured by Illumina

The HiSeq 3000/4000 SBS Kit reagents are a set of laboratory consumables used in the operation of the HiSeq 3000 and HiSeq 4000 sequencing systems manufactured by Illumina. These reagents are designed to enable the sequencing-by-synthesis (SBS) process, which is the core function of the HiSeq platforms.

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Lab products found in correlation

Hiseq 4000, by Illumina (8 mentions) Fragment analyzer, by Agilent Technologies (3 mentions) Sciclone liquid handling robot, by PerkinElmer (3 mentions) Truseq stranded mrna reagents, by Illumina (3 mentions) Hiseq 3000 4000 sr cluster kit reagents, by Illumina (2 mentions) Bcl2fastq2 conversion software, by Illumina (2 mentions) Rnaeasy minielute spin column, by Qiagen (1 mentions) Truseq dna v2, by Illumina (1 mentions) Fragment analyzer, by Thermo Fisher Scientific (1 mentions) Bcl2fastq conversion software, by Illumina (1 mentions) Dnase 1, by Qiagen (1 mentions) Ingenuity pathway analysis (ipa), by Qiagen (1 mentions)

9 protocols using hiseq 3000 4000 sbs kit reagents

Transcriptomic Analysis of Primary Brain Endothelial Cells

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Ch25h^fl/fl and Ch25h^ECKO female mice were injected with tamoxifen. Nine brains per genotype were pooled and primary brain microvascular endothelial cells (pMBMEC) were isolated and plated in a 96‐well plate (2 wells/brain). Confluent pMBMEC were left unstimulated or stimulated IL‐1β for 24 h. RNA of three wells was pooled to obtain one replicate for RNA sequencing. The Lausanne Genomic Technologies Facility performed the RNA‐seq. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies), and all RNAs had a RQN between 8.7 and 10. RNA‐seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies).
Cluster generation was performed with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina).

Ruiz F., Peter B., Rebeaud J., Vigne S., Bressoud V., Roumain M., Wyss T., Yersin Y., Wagner I., Kreutzfeldt M., Pimentel Mendes M., Kowalski C., Boivin G., Roth L., Schwaninger M., Merkler D., Muccioli G.G., Hugues S., Petrova T.V, & Pot C. (2023). Endothelial cell‐derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis. EMBO Reports, 24(3), e55328.

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RNA Extraction and RNA-Seq Analysis in Mice Kidneys

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RNA from frozen half-kidneys of 72 mice were extracted and purified using RNAeasy MiniElute Spin Column (Qiagen). RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). All RNAs had an RNA quality number (RQN) between 7.5 and 9.7. RNA-Seq libraries were prepared from 200 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies). Clusters were generated with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents. Sequencing was performed on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single read). Sequencing data were demultiplexed, filtered for failed reads, and written to FASTQ files using the bcl2fastq2 conversion software (version 2.20, Illumina). Details for RNA-Seq reads mapping are described in Supplemental Methods.

Bignon Y., Wigger L., Ansermet C., Weger B.D., Lagarrigue S., Centeno G., Durussel F., Götz L., Ibberson M., Pradervand S., Quadroni M., Weger M., Amati F., Gachon F, & Firsov D. (2023). Multiomics reveals multilevel control of renal and systemic metabolism by the renal tubular circadian clock. The Journal of Clinical Investigation, 133(8), e167133.

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Illumina mRNA Sequencing Library Prep

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Sequencing libraries were constructed using TruSeq DNA V2 (Illumina, San Diego, CA) sample prep kits and quantified using qPCR (Kapa Biosystems, Wilmington, MA). The mRNA was fragmented, and double-stranded cDNA was generated by random priming. The ends of the fragmented DNA were converted into phosphorylated blunt ends. An ‘A’ base was added to the 3′ ends. Illumina®-specific adaptors were ligated to the DNA fragments. Using magnetic bead technology, the ligated fragments were size-selected and then a final PCR was performed to enrich the adapter-modified DNA fragments, since only the DNA fragments with adaptors at both ends will amplify. Libraries were pooled and sequenced by the Genome Technologies core facility at The Jackson Laboratory. Samples were sequenced on Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents (Illumina), targeting 30 million read pairs per sample. Samples were split across multiple lanes when being run on the Illumina HiSeq, once the data was received the samples were concatenated to have a single file for paired-end analysis.

Preuss C., Pandey R., Piazza E., Fine A., Uyar A., Perumal T., Garceau D., Kotredes K.P., Williams H., Mangravite L.M., Lamb B.T., Oblak A.L., Howell G.R., Sasner M., Logsdon B.A, & Carter G.W. (2020). A novel systems biology approach to evaluate mouse models of late-onset Alzheimer’s disease. Molecular Neurodegeneration, 15, 67.

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High-Throughput Illumina Sequencing Protocol

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Libraries were pooled and sequenced by the Genome Technologies core facility at The Jackson Laboratory. All samples were sequenced on Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents (Illumina), targeting 30 million read pairs per sample. Samples were split across multiple lanes when being run on the Illumina HiSeq, once the data was received the samples were concatenated to have a single file for paired-end analysis.

Kotredes K.P., Oblak A., Pandey R.S., Lin P.B., Garceau D., Williams H., Uyar A., O’Rourke R., O’Rourke S., Ingraham C., Bednarczyk D., Belanger M., Cope Z., Foley K.E., Logsdon B.A., Mangravite L.M., Sukoff Rizzo S.J., Territo P.R., Carter G.W., Sasner M., Lamb B.T, & Howell G.R. (2021). Uncovering Disease Mechanisms in a Novel Mouse Model Expressing Humanized APOEε4 and Trem2*R47H. Frontiers in Aging Neuroscience, 13, 735524.

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RNA Extraction and RNA-seq Library Preparation

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RNA was extracted using the Total RNA Isolation RNeasy Mini Kit with the DNAse I (QIAGEN), on-column digestion step. Snap-frozen pieces of tumor and normal tissue samples (≈30 mg) were directly submerged in 350 µl of RLT buffer (second RNA wash buffer with ethanol) supplemented with 40 µM dithiothreitol. Tissues were completely homogenized on ice using a pestle and passed through a 26G needle syringe 5×. Centrifugation was performed in a table-top centrifuge at 4 °C for 3 min at 18,213g before the supernatant was removed and directly used for RNA extraction.
RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). RNA-seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents using a unique dual indexing strategy and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorimetric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer.
Cluster generation was performed with the resulting libraries using Illumina HiSeq 3000/4000 PE Cluster Kit reagents. Libraries were sequenced on the Illumina HiSeq 4000 with HiSeq 3000/4000 SBS Kit reagents for 2× 150 cycles. Sequencing data were de-multiplexed with the bcl2fastq Conversion Software (v.2.20, Illumina).

Kraemer A.I., Chong C., Huber F., Pak H., Stevenson B.J., Müller M., Michaux J., Altimiras E.R., Rusakiewicz S., Simó-Riudalbas L., Planet E., Wiznerowicz M., Dagher J., Trono D., Coukos G., Tissot S, & Bassani-Sternberg M. (2023). The immunopeptidome landscape associated with T cell infiltration, inflammation and immune editing in lung cancer. Nature Cancer, 4(5), 608-628.

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RNA-seq Analysis of Aging Mice

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Libraries were pooled and sequenced 75 bp single‐end on the HiSeq 4000 (Illumina) using HiSeq 3000/4000 SBS Kit reagents (Illumina), targeting 40 million reads per sample. We obtained RNA‐seq data from spleen, PBL and sorted T cells (derived from spleen) of B6 and NZO mouse strains at age 3, 12, and 18 months. Single‐end RNA‐seq reads were aligned to the mouse genome (mm10) with Bowtie 2 (Langmead & Salzberg, 2012 (link)) and counts were generated with RSEM (Li & Dewey, 2011 (link)). To normalize the raw counts count‐per‐million (cpm) function from edgeR package is used and the genes that are log(cpm) < 1 and expressed less than 2 samples were excluded from rest of the analyses. For differential analysis pipeline, however, raw counts are used with the default options of edgeR package (Robinson et al., 2010 (link)), and via TMM normalization.

Karakaslar E.O., Katiyar N., Hasham M., Youn A., Sharma S., Chung C., Marches R., Korstanje R., Banchereau J, & Ucar D. (2023). Transcriptional activation of Jun and Fos members of the AP‐1 complex is a conserved signature of immune aging that contributes to inflammaging. Aging Cell, 22(4), e13792.

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Illumina HiSeq 4000 Sequencing Protocol

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Libraries were pooled and sequenced by the Genome Technologies core facility at The Jackson Laboratory. All 36 samples were sequenced on an Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents (Illumina), targeting 30 million read pairs per sample. Samples were split across multiple lanes for Illumina HiSeq; once the data were obtained, the samples were concatenated to produce a single file for paired-end analysis.

Oblak A.L., Lin P.B., Kotredes K.P., Pandey R.S., Garceau D., Williams H.M., Uyar A., O’Rourke R., O’Rourke S., Ingraham C., Bednarczyk D., Belanger M., Cope Z.A., Little G.J., Williams S.P., Ash C., Bleckert A., Ragan T., Logsdon B.A., Mangravite L.M., Sukoff Rizzo S.J., Territo P.R., Carter G.W., Howell G.R., Sasner M, & Lamb B.T. (2021). Comprehensive Evaluation of the 5XFAD Mouse Model for Preclinical Testing Applications: A MODEL-AD Study. Frontiers in Aging Neuroscience, 13, 713726.

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RNA-seq Analysis of Immune Cell Responses

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For the RNA-seq of BM-DCs, total RNA was extracted from BM-DCs after treatment with WT or Ptgs2^−/− lung fibroblast CM, or PGE2 (10 ng/ml) for 16 hours. For the RNA-seq of lung fibroblasts, total RNA was extracted from fibroblasts after treatment with IL-1β (10 ng/ml) for 24 hours. For the RNA-seq of lung myeloid cells, CD11b⁺ DCs, CD103⁺ DCs and conventional monocytes were sorted from WT or Ptgs2^ΔFb naïve mice. Total RNA isolation, sample quality check and library preparation were performed by the Genome Technologies core facility at The Jackson Laboratory. After the quality check, libraries were pooled and sequenced, 100-bp paired-end on the HiSeq 4000 (Illumina) by using HiSeq 3000/4000 SBS kit reagents (Illumina). Paired-end reads were aligned to Mus musculus reference GRCm38. The transcripts per million (TPM) results of RNA-seq data were used in the limma package (version 3.46.0) (Ritchie et al., 2015 (link)) to determine differentially expressed genes. Ingenuity Pathway Analysis (QIAGEN) was used to predict candidate upstream regulators.

Gong Z., Li Q., Shi J., Wei J., Li P., Chang C.H., Shultz L.D, & Ren G. (2022). Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment. Immunity, 55(8), 1483-1500.e9.

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Genome Sequencing on Illumina HiSeq 4000

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Sasner M., Preuss C., Pandey R.S., Uyar A., Garceau D., Kotredes K.P., Williams H., Oblak A.L., Lin P.B., Perkins B., Soni D., Ingraham C., Lee-Gosselin A., Lamb B.T., Howell G.R, & Carter G.W. (2023). In vivo validation of late-onset Alzheimer’s disease genetic risk factors. bioRxiv.

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