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Hiseq v4 cluster and sbs kits

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The HiSeq v4 Cluster and SBS kits are laboratory equipment used for next-generation sequencing. The Cluster kit is responsible for preparing DNA samples for sequencing, while the SBS (Sequencing by Synthesis) kit is used to perform the actual sequencing reaction. These kits are designed to work with the HiSeq sequencing platform manufactured by Illumina.

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Lab products found in correlation

Hiseq 2500 sequencer, by Illumina (5 mentions) Qubit fluorometer, by Thermo Fisher Scientific (5 mentions) Nextera xt dna sample preparation kit, by Illumina (4 mentions) Smart seq v4 ultra low input, by Takara Bio (2 mentions) Smart seq v4, by Takara Bio (1 mentions) Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (1 mentions) Basespace, by Illumina (1 mentions) Truseq stranded mrna kit, by Illumina (1 mentions)

Spelling variants of this product

HiSeq SBS Kit v4 (63 citations) V4 SBS and Cluster Kits (4 citations) HiSeq Cluster kit v4 (3 citations) SBS kits (3 citations)

5 protocols using hiseq v4 cluster and sbs kits

1

Single-cell RNA-seq library preparation

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Single cells were sorted directly into SMARTseq v4 (Takara) lysis buffer to release RNA. Reverse transcription was performed followed by PCR amplification to generate full length amplified cDNA. Sequencing libraries were constructed using a modified protocol of the NexteraXT DNA sample preparation kit (Illumina) to generate Illumina-compatible barcoded libraries. Libraries were pooled and quantified using a Qubit® Fluorometer (Life Technologies). Dual-index, single-read sequencing of the pooled libraries was carried out on a HiSeq2500 sequencer (Illumina) with 58-base reads, using HiSeq v4 Cluster and SBS kits (Illumina) with a target depth of 1 million reads per sample. FASTQs were aligned to a human reference genome to generate gene counts.
Menard L.C., Fischer P., Kakrecha B., Linsley P.S., Wambre E., Liu M.C., Rust B.J., Lee D., Penhallow B., Manjarrez Orduno N, & Nadler S.G. (2018). Renal Cell Carcinoma (RCC) Tumors Display Large Expansion of Double Positive (DP) CD4+CD8+ T Cells With Expression of Exhaustion Markers. Frontiers in Immunology, 9, 2728.
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2

Single-cell RNA-seq of CD4+ T cells

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We performed bulk RNA-sequencing on 110–200 sorted CD4Tconv and Treg from the dLN of infected and uninfected mice and the VT of infected mice. Cells were not pooled between mice. 11 samples were sequenced: 4 CD4+FoxP3+ from infected dLN; 4 CD4+FoxP3+ from infected VT; 3 CD4+FoxP3+ from uninfected dLN. Briefly, as previously described89 , cells were sorted into SMART-seq v4 Ultra Low Input (Takara Bio USA, San Jose, California) lysis buffer and reverse transcription was performed followed by PCR amplification to generate full length amplified cDNA. Sequencing libraries were constructed using the NexteraXT DNA sample preparation kit (Illumina) to generate Illumina-compatible barcoded libraries. Libraries were pooled and quantified using a Qubit® Fluorometer (Life Technologies). Dual-index, single-read sequencing of the pooled libraries was carried out on a HiSeq2500 sequencer (Illumina) with 58-base reads, using HiSeq v4 Cluster and SBS kits (Illumina) with a target depth of 5 million reads per sample. FASTQs were aligned to a mouse reference genome, using STAR v.2.4.2a and gene counts were generated using htseq-count. QC and metrics analysis was performed using the Picard family of tools (v1.134).
Traxinger B., Vick S.C., Woodward-Davis A., Voillet V., Erickson J.R., Czartoski J., Teague C., Prlic M, & Lund J.M. (2022). Mucosal viral infection induces a regulatory T cell activation phenotype distinct from tissue residency in mouse and human tissues. Mucosal immunology, 15(5), 1012-1027.
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3

Single-cell mRNA-Seq Library Preparation

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Total RNA was added directly to lysis buffer from the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara), and reverse transcription was performed followed by PCR amplification to generate full-length amplified cDNA. Sequencing libraries were constructed using the NexteraXT DNA sample preparation kit (Illumina) to generate Illumina-compatible barcoded libraries. Libraries were pooled and quantified using a Qubit® Fluorometer (Life Technologies). Dual-index, single-read sequencing of pooled libraries was carried out on a HiSeq2500 sequencer (Illumina) with 58-base reads, using HiSeq v4 Cluster and SBS kits (Illumina) with a target depth of 5 million reads per sample. Details of mRNA-Seq analyses and software are listed in the Supplementary Materials and Methods.
Burleigh K., Maltbaek J.H., Cambier S., Green R., Gale M., James R.C, & Stetson D.B. (2020). Human DNA-PK activates a STING-independent DNA sensing pathway. Science immunology, 5(43), eaba4219.
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4

RNA-sequencing of Human Samples

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RNA-seq was performed by the Genomics Core at the Benaroya Research Institute. For each sample, libraries were constructed from 100 ng of total RNA using the TruSeq Stranded mRNA kit (Illumina) with poly(A) selection. Libraries were pooled and quantified using a Qubit® Fluorometer (Life Technologies). Single-read sequencing of pooled libraries was carried out on a HiSeq2500 sequencer (Illumina) with 58-base reads, using HiSeq v4 Cluster and SBS kits (Illumina) with a target depth of 10 million reads per sample. Basecalls were processed to FASTQs on BaseSpace (Illumina), and a base call quality trimming step was applied to remove low-confidence base calls from the ends of reads. The FASTQs were aligned to the human reference genome (GRCh38.91), using STAR v.2.4.2a (Dobin et al., 2013 (link)) and gene counts were generated using htseq-count (Anders et al., 2015 (link)). QC and metrics analysis were performed using the Picard family of tools (v1.134) (https://broadinstitute.github.io/picard/). All RNA-seq data meeting MINSEQE (Minimum Information About a Next-generation Sequencing Experiment) have been deposited at Gene Expression Omnibus repository (https://www.ncbi.nlm.nih.gov/geo/, GSE148076).
Hisert K.B., Birkland T.P., Schoenfelt K.Q., Long M.E., Grogan B., Carter S., Liles W.C., McKone E.F., Becker L., Manicone A.M, & Gharib S.A. (2020). CFTR Modulator Therapy Enhances Peripheral Blood Monocyte Contributions to Immune Responses in People With Cystic Fibrosis. Frontiers in Pharmacology, 11, 1219.
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5

Single-cell RNA-seq of CD4+ T cells

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We performed bulk RNA-sequencing on 110–200 sorted CD4Tconv and Treg from the dLN of infected and uninfected mice and the VT of infected mice. Cells were not pooled between mice. 11 samples were sequenced: 4 CD4+FoxP3+ from infected dLN; 4 CD4+FoxP3+ from infected VT; 3 CD4+FoxP3+ from uninfected dLN. Briefly, as previously described89 , cells were sorted into SMART-seq v4 Ultra Low Input (Takara Bio USA, San Jose, California) lysis buffer and reverse transcription was performed followed by PCR amplification to generate full length amplified cDNA. Sequencing libraries were constructed using the NexteraXT DNA sample preparation kit (Illumina) to generate Illumina-compatible barcoded libraries. Libraries were pooled and quantified using a Qubit® Fluorometer (Life Technologies). Dual-index, single-read sequencing of the pooled libraries was carried out on a HiSeq2500 sequencer (Illumina) with 58-base reads, using HiSeq v4 Cluster and SBS kits (Illumina) with a target depth of 5 million reads per sample. FASTQs were aligned to a mouse reference genome, using STAR v.2.4.2a and gene counts were generated using htseq-count. QC and metrics analysis was performed using the Picard family of tools (v1.134).
Traxinger B., Vick S.C., Woodward-Davis A., Voillet V., Erickson J.R., Czartoski J., Teague C., Prlic M, & Lund J.M. (2022). Mucosal viral infection induces a regulatory T cell activation phenotype distinct from tissue residency in mouse and human tissues. Mucosal immunology, 15(5), 1012-1027.
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