Hiseq sr cluster kit v4 cbot

Manufactured by Illumina

The HiSeq SR Cluster Kit v4 cBot is a laboratory equipment product designed for use with the cBot system. It is used for the generation of DNA clusters on flow cells, which is a necessary step in the sequencing process on Illumina's HiSeq platform. The kit provides reagents and materials required to prepare samples and perform this cluster generation step.

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Lab products found in correlation

Hiseq sbs kit v4, by Illumina (3 mentions) Hiseq 2500 instrument, by Illumina (3 mentions) Hiseq v4 single read flow cell, by Illumina (3 mentions) Hiseq sbs kit v4 sequencing reagents, by Illumina (3 mentions) Truseq stranded mrna library preparation kit, by Illumina (2 mentions) D1000 screentape assay, by Agilent Technologies (2 mentions) Tapestation 2200, by Agilent Technologies (2 mentions) Rneasy mini kit, by Qiagen (2 mentions) Hiseqv4, by Illumina (2 mentions) Hiseq 2500, by Illumina (2 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Hiseq 2500 system, by Illumina (1 mentions) Truseq rna sample preparation kit v2, by Illumina (1 mentions) Cbot system, by Illumina (1 mentions) Kapa library quant kit, by Roche (1 mentions) Truseq stranded total rna sample prep, by Illumina (1 mentions) Truseq stranded mrna sample preparation kit, by Illumina (1 mentions) Truseq rna library prep kit v2, by Illumina (1 mentions) Quant it ribogreen rna assay kit, by Agilent Technologies (1 mentions) Fragment analyzer, by Illumina (1 mentions)

9 protocols using hiseq sr cluster kit v4 cbot

RNA-sequencing library preparation and analysis

Cited 4 times

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We converted 150 ng of total RNA to cDNA using Superscript II reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). Then, sequencing libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 (Illumina). Library quality was assessed as previously described.^{65 (link)} For cluster generation with the HiSeq SR Cluster Kit v4 cBot (Illumina), six libraries were mixed in equimolar concentrations and were loaded into flow cells. Sequencing was performed in the HiSeq 2500 system (Illumina) with the HiSeq SBS Kit v4 (single-end 125-bp reads).
Read sequences were mapped onto the GRCh37 human reference genome using TopHat^{66 (link)} v2.0.13 and a guide from the Human GENCODE Gene Set (release 19).^{54 (link)} We removed reads mapped to transfer RNA and ribosomal RNA regions. Multi-mapped reads and reads with mapping quality <50 were excluded. Fragments per kb of exon per million mapped fragments values were calculated and normalised across subjects using the cuffquant and cuffnorm programs in the Cufflinks package^{67 (link)} v2.2.1.

Hachiya T., Furukawa R., Shiwa Y., Ohmomo H., Ono K., Katsuoka F., Nagasaki M., Yasuda J., Fuse N., Kinoshita K., Yamamoto M., Tanno K., Satoh M., Endo R., Sasaki M., Sakata K., Kobayashi S., Ogasawara K., Hitomi J., Sobue K, & Shimizu A. (2017). Genome-wide identification of inter-individually variable DNA methylation sites improves the efficacy of epigenetic association studies. NPJ Genomic Medicine, 2, 11.

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RNA-seq protocol for IFN-stimulated MDM

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MDM were generated as described and stimulated with 25 ng/ml of the indicated IFNs. Total RNA was isolated 18 h following stimulation using RNeasy minikit (Qiagen). Intact poly(A) RNA was purified from total RNA samples (100 to 500 ng) with oligo(dT) magnetic beads, and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq Stranded mRNA Library Preparation kit (catalog no. RS-122-2101 and RS-122-2102). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (catalog no. 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Biosystems Kapa Library Quant kit (catalog no. KK4824). Individual libraries were normalized to 10 nM, and equal volumes were pooled in preparation for Illumina sequence analysis. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot system. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR cluster kit v4-cBot (catalog no. GD-401-4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS kit v4 sequencing reagents (catalog no. FC-401-4002).

Szaniawski M.A., Spivak A.M., Cox J.E., Catrow J.L., Hanley T., Williams E.S., Tremblay M.J., Bosque A, & Planelles V. (2018). SAMHD1 Phosphorylation Coordinates the Anti-HIV-1 Response by Diverse Interferons and Tyrosine Kinase Inhibition. mBio, 9(3), e00819-18.

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Transcriptome Profiling with Illumina HiSeq

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Illumina TruSeq Stranded Total RNA Sample Prep with Ribo-Zero was used to prepare the library. Libraries were chemically denatured and applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot. After transfer of the flow cell to an Illumina HiSeq 2500, a 50-cycle single-read sequence run was performed (HiSeq SBS Kit v4). For data analysis, Rn5 Ensembl annotations (Build 75) were downloaded and converted to genePred format. Reads were aligned to the transcriptome reference index using NovoAlign (v2.08.01), allowing up to 50 alignments for each read. Read counts were generated using the USeq Defined Region Differential Seq application and used in DESeq2 to measure the differential expression between each condition, controlling for sample preparation batch. Data has been submitted to the GEO Database (Accession # GSE110804). For Ingenuity Pathway Analysis (IPA), differentially expressed gene lists were used as input to identify related pathways, diseases, and networks.

Pan H., Strickland A., Madhu V., Johnson Z.I., Chand S.N., Brody J.R., Fertala A., Zheng Z., Shapiro I.M, & Risbud M.V. (2018). RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells. Matrix biology : journal of the International Society for Matrix Biology, 77, 23-40.

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RNA Isolation and Sequencing of Mouse Tumors

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RNA isolation from ~15 mg flash-frozen RPM (n = 10) and RPR2 (n = 5) primary tumors from mice infected with Ad5-CGRP-Cre or flash-frozen RPM transition cell pellets (n = 8: Days 3, 5, 7, 10, 12, 14, 19, 21) was performed using RNeasy Mini Kit (Qiagen) with the standard protocol. RNA from RPM tumors (n = 10) was subject to library construction with the Illumina TruSeq Stranded mRNA Sample Preparation Kit (cat# RS-122-2101, RS-122-2102) according to the manufacturer’s protocol. RNA from RPR2 (n = 5) tumors and RPM transition timepoints (n = 8) were subject to library construction with the Illumina TruSeq Stranded Total RNA Library Ribo-Zero Gold Prep kit (cat# RS-122-2301) according to the manufacturer’s protocol. Chemically denatured sequencing libraries (25 pM) from RPM (n = 10) and RPR2 (n = 5) tumors and RPM transition timepoints (n = 8) were applied to an Illumina HiSeq v4 single read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD-401-4001). Following transfer of the flowcell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-end sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC-401-4002).

Ireland A.S., Micinski A.M., Kastner D.W., Guo B., Wait S.J., Spainhower K.B., Conley C.C., Chen O.S., Guthrie M.R., Soltero D., Qiao Y., Huang X., Tarapcsak S., Devarakonda S., Chalishazar M.D., Gertz J., Moser J.C., Marth G., Puri S., Witt B.L., Spike B.T, & Oliver T.G. (2020). MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate. Cancer cell, 38(1), 60-78.e12.

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Temporal profiling of neural cell types

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Total RNA was isolated from FAC‐sorted GFP‐positive cells from Hes5::GFP and Tbr2::GFP transgenic lines using TRIzol reagent. A time course was performed with NSCs, BPs, and NBNs isolated at each time point during development from E10.5 to postnatal day 1 (PN) or as specified in the Fig 1A. Samples were analyzed for their integrity and concentration using Agilent 2100 Bioanalyzer and Quant‐IT RiboGreen RNA Assay Kit. Sequencing libraries were prepared with the Illumina TruSeq RNA Library Prep Kit v2 according to Illumina's instructions. After quality control (Fragment Analyzer, AATI), libraries were pooled and loaded on an Illumina flow cell for cluster generation (HiSeq SR Cluster Kit v4 cBot). Libraries were sequenced SR50 on the HiSeq 2500 system (HiSeq SBS Kit V4) following the manufacturer's protocols.

Mukhtar T., Breda J., Adam M.A., Boareto M., Grobecker P., Karimaddini Z., Grison A., Eschbach K., Chandrasekhar R., Vermeul S., Okoniewski M., Pachkov M., Harwell C.C., Atanasoski S., Beisel C., Iber D., van Nimwegen E, & Taylor V. (2022). Temporal and sequential transcriptional dynamics define lineage shifts in corticogenesis. The EMBO Journal, 41(24), e111132.

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RNA-seq Analysis of Cultured Cells

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Total RNA from cultured cells was extracted using TRIzol reagent (Invitrogen). Strand-specific libraries were generated using the TruSeq PolyA Stranded mRNA sample preparation kit (iIlumina). In brief, polyadenylated RNA was purified using oligo-dT beads. Following purification, the RNA was fragmented, random-primed and reserve transcribed using SuperScript II Reverse Transcriptase (Invitrogen). The generated cDNA was 3′ end-adenylated and ligated to Illumina Paired-end sequencing adapters and amplified by PCR using HiSeq SR Cluster Kit v4 cBot (Illumina). Libraries were analyzed on a 2100 Bioanalyzer (Agilent) and subsequently sequenced on a HiSeq2000 (Illumina). We performed RNaseq alignment using TopHat 2.1.1 (Kim et al., 2013 (link)). Differentially expressed genes were called with DEseq2 (Love et al., 2014 (link)), with an adjusted p value threshold of 0.05.

Haarhuis J.H., van der Weide R.H., Blomen V.A., Yáñez-Cuna J.O., Amendola M., van Ruiten M.S., Krijger P.H., Teunissen H., Medema R.H., van Steensel B., Brummelkamp T.R., de Wit E, & Rowland B.D. (2017). The Cohesin Release Factor WAPL Restricts Chromatin Loop Extension. Cell, 169(4), 693-707.e14.

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Ribosome Profiling of Platelets

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For ribosome footprint profiling experiments, 1 × 10⁹ platelets were treated with cycloheximide (100 mg/ml) for 1 min at RT to preserve ribosomes as natively attached to mRNAs.^{35 (link)} The platelets were lysed in Mammalian Lysis Buffer (ARTseq, Epicentre). Total RNA and ribosome-protected read RNAs (RPRs) were prepared per the manufacturer’s instructions (ARTseq-R ibosome Profiling Kit, Epicentre). Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR Cluster Kit v4-cBot (GD- 401– 4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCSv2.2.38 and RTA v1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS Kit v4 sequencing reagents (FC- 401– 4002). Ribosome profiling (eukaryote) library preparation was completed using the NEBNext Multiplex Small RNA Library Prep Set.

Eustes A.S., Campbell R.A., Middleton E.A., Tolley N.D., Manne B.K., Montenont E., Rowley J.W., Krauel K., Blair A., Guo L., Kosaka Y., Medeiros- de- Moraes I.M., Lacerda M., Hottz E.D., Neto H.C., Zimmerman G.A., Weyrich A.S., Petrey A, & Rondina M.T. (2021). Heparanase expression and activity are increased in platelets during clinical sepsis. Journal of thrombosis and haemostasis : JTH, 19(5), 1319-1330.

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Single-cell RNA-seq from Sorted Cells

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Total RNA was isolated from sorted cell populations using an miRNeasy Micro kit (Qiagen) and quantified. Three nanograms of total RNA were used to generate cDNA following the Smart-seq2 protocol. cDNA was purified using AMPure XP beads (0.8×, Beckman Coulter). Next, 1 ng of cDNA from each sample was used to generate a sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). The libraries were pooled and sequenced on a HiSeq 2500 (Illumina) to obtain 50-bp single-end reads. Both full-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality control steps were included after each step to eliminate samples with low quality from the downstream process. A detailed protocol has been previously published (Rosales et al. 2018 (link)). Libraries were sequenced on a HiSeq 2500 Illumina to obtain a minimum of 10 million 50-bp single-end reads (HiSeq SR Cluster kit v4 cBot, HiSeq SBS kit v4).

Panwar B., Schmiedel B.J., Liang S., White B., Rodriguez E., Kalunian K., McKnight A.J., Soloff R., Seumois G., Vijayanand P, & Ay F. (2021). Multi–cell type gene coexpression network analysis reveals coordinated interferon response and cross–cell type correlations in systemic lupus erythematosus. Genome Research, 31(4), 659-676.

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Illumina Stranded mRNA Sequencing Library Preparation

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Intact poly(A) RNA was purified from total RNA samples (100–500 ng) with oligo(dT) magnetic beads, and stranded mRNA sequencing libraries were prepared as described using the Illumina TruSeq stranded mRNA library preparation kit (RS-122-2101 and RS-122-2102). Purified libraries were qualified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay (catalog nos. 5067-5582 and 5067-5583). The molarity of adapter-modified molecules was defined by qPCR using the Kapa Biosystems Kapa library quantification kit (KK4824). Individual libraries were normalized to 10 nM, and equal volumes were pooled in preparation for Illumina sequence analysis.
Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq v4 single-read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina HiSeq SR cluster kit v4-cBot (GD-401-4001). Following transfer of the flow cell to an Illumina HiSeq 2500 instrument (HCS version 2.2.38 and RTA version 1.18.61), a 50-cycle single-read sequence run was performed using HiSeq SBS kit version 4 sequencing reagents (FC-401-4002).

Pearson S., Loft A., Rajbhandari P., Simcox J., Lee S., Tontonoz P., Mandrup S, & Villanueva C.J. (2019). Loss of TLE3 promotes the mitochondrial program in beige adipocytes and improves glucose metabolism. Genes & Development, 33(13-14), 747-762.

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