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Cbot and hiseq 3000 4000 pe cluster kit

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The CBot and HiSeq 3000/4000 PE Cluster Kit is a set of laboratory equipment and reagents designed for the automated generation of clustered DNA samples for use in next-generation sequencing workflows on the Illumina HiSeq 3000 and HiSeq 4000 platforms. The core function of this product is to prepare DNA samples for sequencing by generating dense clusters of clonally amplified DNA fragments on flow cells.

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Lab products found in correlation

Hiseq 4000, by Illumina (6 mentions) Hiseq 3000 4000 sequencing kit, by Illumina (5 mentions) Qubit fluorometry, by Thermo Fisher Scientific (4 mentions) Rta version 2, by Illumina (4 mentions) Bioanalyzer dna 1000 chip, by Thermo Fisher Scientific (2 mentions) Rta software version 2, by Illumina (2 mentions) Rneasy plus mini kit, by Qiagen (2 mentions) Truseq rna sample prep kit v2, by Illumina (2 mentions) Paxgene blood rna tube, by Qiagen (2 mentions) Truseq rna access library prep kit, by Illumina (2 mentions) Mirneasy mini kit, by Qiagen (2 mentions) Vi cell xr cell viability analyzer, by Beckman Coulter (1 mentions) Chromium single cell 3 reagent kit, by 10x Genomics (1 mentions) Cell ranger v3, by 10x Genomics (1 mentions) Ribo zero gold, by Illumina (1 mentions) Rta v2, by Illumina (1 mentions) Truseq stranded total rna sample prep kit, by Illumina (1 mentions) Hiseq 4000 instrument, by Illumina (1 mentions) Agilent bravo, by Agilent Technologies (1 mentions) Sureselect human all exon v5 utrs 75 mb kit, by Agilent Technologies (1 mentions)

8 protocols using cbot and hiseq 3000 4000 pe cluster kit

1

Single-cell RNA-seq Analysis Pipeline

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Single-cell suspensions were counted using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter, Brea, CA, USA) and ~5000 cells were loaded into the 10X Genomics Chromium system to generate Gel Beads-In-Emulsion (GEMs). Reverse transcription was performed using Chromium Single Cell 3’ Reagent Kits (v2, 10X Genomics, Pleasanton, CA, USA). Standard Illumina sequencing primers and a unique i7 Sample index were added to each cDNA for library construction. Libraries were sequenced using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3. Cell Ranger (v3.0) (10X Genomics) was used for alignment to the mouse genome version mm10. R (v3.6.0) and Seurat (v3.1.3)35 (link) were used for further analysis following the standard workflow. For quality control and filtering, cells with detected genes fewer than 200 or >8000, or with mitochondrial gene content >50% were excluded. For Ximerakis’s scRNA-seq dataset, R (v4.0.3) and Seurat (v4.0.2) were used. Gene set enrichment analysis was performed using software GSEA (v4.0.3)53 (link). The interactive website for scRNA-seq data was generated using ShinyCell (v2.1.0)54 .
Zhang X., Pearsall V.M., Carver C.M., Atkinson E.J., Clarkson B.D., Grund E.M., Baez-Faria M., Pavelko K.D., Kachergus J.M., White T.A., Johnson R.K., Malo C.S., Gonzalez-Suarez A.M., Ayasoufi K., Johnson K.O., Tritz Z.P., Fain C.E., Khadka R.H., Ogrodnik M., Jurk D., Zhu Y., Tchkonia T., Revzin A., Kirkland J.L., Johnson A.J., Howe C.L., Thompson E.A., LeBrasseur N.K, & Schafer M.J. (2022). Rejuvenation of the aged brain immune cell landscape in mice through p16-positive senescent cell clearance. Nature Communications, 13, 5671.
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2

Bulk RNA-seq and Single Myofiber Library Preparation

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For bulk RNA-seq, cDNA libraries were prepared according to the manufacturer’s instructions for the TruSeq Stranded mRNA Sample Prep Kit for the muscle tissue (Illumina, San Diego, CA) or SMART-Seq v4 Ultra Low Input RNA Kit for the single myofiber (Clontech). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit on an Illumina HiSeq 4000.
Zhang X., Habiballa L., Aversa Z., Ng Y.E., Sakamoto A.E., Englund D.A., Pearsall V.M., White T.A., Robinson M.M., Rivas D.A., Dasari S., Hruby A.J., Lagnado A.B., Jachim S.K., Granic A., Sayer A.A., Jurk D., Lanza I.R., Khosla S., Fielding R.A., Nair K.S., Schafer M.J., Passos J.F, & LeBrasseur N.K. (2022). Characterization of cellular senescence in aging skeletal muscle. Nature aging, 2(7), 601-615.
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3

RNA-seq Analysis of Mouse Alveolar Macrophages

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Total RNA from in vitro cultured AMs was extracted using RNeasy Plus Mini Kit (QIAGEN) following the manufacture’s protocol. Two pools per genotype were used for RNA-seq. After quality control, high quality (Agilent Bioanalyzer RIN > 7.0) total RNA was used to generate the RNA sequencing library. cDNA synthesis, end-repair, A-base addition, and ligation of the Illumina indexed adapters were performed according to the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). The concentration and size distribution of the completed libraries was determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Paired-end libraries were sequenced on an Illumina HiSeq 4000 following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. Base-calling was performed using Illumina’s RTA software (version 2.5.2). Paired-end RNA-seq reads were aligned to the mouse reference genome (GRCm38/mm10) using Bowtie (v2.3.4). Pre- and post-alignment quality controls, gene level raw read count and normalized read count (i.e., FPKM) were performed using RSeQC package (v2.3.6) with NCBI mouse RefSeq gene model. Differential expression for each gene between various groups specified in the text was identified by Cuffdiff (Trapnell et al., 2010 (link)).
Zhu B., Wu Y., Huang S., Zhang R., Son Y.M., Li C., Cheon I.S., Gao X., Wang M., Chen Y., Zhou X., Nguyen Q., Phan A.T., Behl S., Taketo M.M., Mack M., Shapiro V.S., Zeng H., Ebihara H., Mullon J.J., Edell E.S., Reisenauer J.S., Demirel N., Kern R.M., Chakraborty R., Cui W., Kaplan M.H., Zhou X., Goldrath A.W, & Sun J. (2021). Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection. Immunity, 54(6), 1200-1218.e9.
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4

RNA-Seq Protocol for Alveolar Macrophages

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Total RNA from in vitro cultured AMs was extracted using RNeasy Plus Mini Kit (Qiagen) following the manufacture’s protocol. Two pools per genotype were used for RNA-seq. After quality control, high quality (Agilent Bioanalyzer RIN >7.0) total RNA was used to generate the RNA sequencing library. cDNA synthesis, end-repair, A-base addition, and ligation of the Illumina indexed adapters were performed according to the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). The concentration and size distribution of the completed libraries was determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Paired-end libraries were sequenced on an Illumina HiSeq 4000 following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. Base-calling was performed using Illumina’s RTA software (version 2.5.2). Paired-end RNA-seq reads were aligned to the mouse reference genome (GRCm38/mm10) using Bowtie (v2.3.4). Pre- and post-alignment quality controls, gene level raw read count and normalized read count (i.e. FPKM) were performed using RSeQC package (v2.3.6) with NCBI mouse RefSeq gene model. Differential expression for each gene between various groups specified in the text was identified by Cuffdiff (Trapnell et al., 2010 (link)).
Zhu B., Wu Y., Huang S., Zhang R., Son Y.M., Li C., Cheon I.S., Gao X., Wang M., Chen Y., Zhou X., Nguyen Q., Phan A.T., Behl S., Taketo M.M., Mack M., Shapiro V.S., Zeng H., Ebihara H., Mullon J.J., Edell E.S., Reisenauer J.S., Demirel N., Kern R.M., Chakraborty R., Cui W., Kaplan M.H., Zhou X., Goldrath A.W, & Sun J. (2021). Uncoupling of macrophage inflammation from self-renewal modulates host recovery from respiratory viral infection. Immunity, 54(6), 1200-1218.e9.
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5

SARS-CoV-2 Infection of hiPSC-Derived Cardiomyocytes

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The hiPSC CMs were infected with SARS-CoV-2/UW001/Human/2020/Wisconsin (UW-001) at an MOI of 0.01. Cells were lysed in TRIzol and were kept at −80°C. Total RNA of the lysate was extracted using a Direct-zol RNA miniprep kit (R2050). Library preparation and sequencing were performed at the Mayo Clinic Genome Analysis Core (GAC).
Briefly, cDNA libraries were prepared using 100 ng of total RNA according to the manufacturer’s instructions for the Illumina TruSeq stranded total RNA sample prep kit with Ribo-Zero Gold (Illumina, San Diego, CA). The concentration and size distribution of the completed libraries were determined using an Agilent Bioanalyzer DNA 1000 chip (Santa Clara, CA) and Qubit fluorometry (Invitrogen, Carlsbad, CA). Libraries were sequenced at three samples per lane to generate approximately 119 to 137 million fragment reads per sample following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE cluster kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 instrument using a HiSeq 3000/4000 sequencing kit and HD 3.4.0.38 collection software. Base-calling was performed using Illumina’s RTA v2.7.7.
Navaratnarajah C.K., Pease D.R., Halfmann P.J., Taye B., Barkhymer A., Howell K.G., Charlesworth J.E., Christensen T.A., Kawaoka Y., Cattaneo R, & Schneider J.W. (2021). Highly Efficient SARS-CoV-2 Infection of Human Cardiomyocytes: Spike Protein-Mediated Cell Fusion and Its Inhibition. Journal of Virology, 95(24), e01368-21.
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6

RNA-seq Analysis of P27 Variant

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RNA sequencing was performed from blood RNA from P27 bearing the p.(Trp1787*) variant by first isolating RNA using the miRNeasy Mini Kit (Qiagen) following the standard protocol from blood drawn in a PAXgene Blood RNA Tube (Qiagen). RNA libraries were prepared, and coding regions of the transcriptome were captured by pooling four of the cDNA libraries at 200 ng each according to the manufacturer’s instructions for the TruSeq® RNA Access Library Prep Kit (Illumina)68 (link). Libraries were sequenced at ~65 million fragment reads per sample (4 samples/lane) following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2. RNA-sequencing analysis was performed using MAP-RSeq69 (link). Reads were aligned to the human genome (hg19) and transcriptome using Tophat270 (link) running Bowtie (v1)71 (link). Gene and exon level read counts were generated using HiSeq72 (link) and BedTools73 (link), respectively. Alignments were visualized using Integrative Genomics Viewer (IGV) (http://software.broadinstitute.org/software/igv/).
Cousin M.A., Creighton B.A., Breau K.A., Spillmann R.C., Torti E., Dontu S., Tripathi S., Ajit D., Edwards R.J., Afriyie S., Bay J.C., Harper K.M., Beltran A.A., Munoz L.J., Rodriguez L.F., Stankewich M.C., Person R.E., Si Y., Normand E.A., Blevins A., May A.S., Bier L., Aggarwal V., Mancini G.M., van Slegtenhorst M.A., Cremer K., Becker J., Engels H., Aretz S., MacKenzie J.J., Brilstra E., van Gassen K.L., van Jaarsveld R.H., Oegema R., Parsons G.M., Mark P., Helbig I., McKeown S.E., Stratton R., Cogne B., Isidor B., Cacheiro P., Smedley D., Firth H.V., Bierhals T., Kloth K., Weiss D., Fairley C., Shieh J.T., Kritzer A., Jayakar P., Kurtz-Nelson E., Bernier R.A., Wang T., Eichler E.E., van de Laar I.M., McConkie-Rosell A., McDonald M.T., Kemppainen J., Lanpher B.C., Schultz-Rogers L.E., Gunderson L.B., Pichurin P.N., Yoon G., Zech M., Jech R., Winkelmann J., Beltran A.S., Zimmermann M.T., Temple B., Moy S.S., Klee E.W., Tan Q.K, & Lorenzo D.N. (2021). Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome. Nature genetics, 53(7), 1006-1021.
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7

RNA-seq Analysis of P27 Variant

Cited 2 times
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RNA sequencing was performed from blood RNA from P27 bearing the p.(Trp1787*) variant by first isolating RNA using the miRNeasy Mini Kit (Qiagen) following the standard protocol from blood drawn in a PAXgene Blood RNA Tube (Qiagen). RNA libraries were prepared, and coding regions of the transcriptome were captured by pooling four of the cDNA libraries at 200 ng each according to the manufacturer’s instructions for the TruSeq® RNA Access Library Prep Kit (Illumina)68 (link). Libraries were sequenced at ~65 million fragment reads per sample (4 samples/lane) following Illumina’s standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.20 collection software. Base-calling was performed using Illumina’s RTA version 2.5.2. RNA-sequencing analysis was performed using MAP-RSeq69 (link). Reads were aligned to the human genome (hg19) and transcriptome using Tophat270 (link) running Bowtie (v1)71 (link). Gene and exon level read counts were generated using HiSeq72 (link) and BedTools73 (link), respectively. Alignments were visualized using Integrative Genomics Viewer (IGV) (http://software.broadinstitute.org/software/igv/).
Cousin M.A., Creighton B.A., Breau K.A., Spillmann R.C., Torti E., Dontu S., Tripathi S., Ajit D., Edwards R.J., Afriyie S., Bay J.C., Harper K.M., Beltran A.A., Munoz L.J., Rodriguez L.F., Stankewich M.C., Person R.E., Si Y., Normand E.A., Blevins A., May A.S., Bier L., Aggarwal V., Mancini G.M., van Slegtenhorst M.A., Cremer K., Becker J., Engels H., Aretz S., MacKenzie J.J., Brilstra E., van Gassen K.L., van Jaarsveld R.H., Oegema R., Parsons G.M., Mark P., Helbig I., McKeown S.E., Stratton R., Cogne B., Isidor B., Cacheiro P., Smedley D., Firth H.V., Bierhals T., Kloth K., Weiss D., Fairley C., Shieh J.T., Kritzer A., Jayakar P., Kurtz-Nelson E., Bernier R.A., Wang T., Eichler E.E., van de Laar I.M., McConkie-Rosell A., McDonald M.T., Kemppainen J., Lanpher B.C., Schultz-Rogers L.E., Gunderson L.B., Pichurin P.N., Yoon G., Zech M., Jech R., Winkelmann J., Beltran A.S., Zimmermann M.T., Temple B., Moy S.S., Klee E.W., Tan Q.K, & Lorenzo D.N. (2021). Pathogenic SPTBN1 variants cause an autosomal dominant neurodevelopmental syndrome. Nature genetics, 53(7), 1006-1021.
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8

Trio Whole Exome Sequencing Analysis

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WES from the second miscarriage and both parents was performed at the Clinical Genomics Laboratory (Mayo Clinic). Paired-end libraries were prepared using 1.0 µg of genomic DNA using the Agilent Bravo liquid handler (Agilent) as indicated by the manufacturer. Whole exon capture was carried out using 750 ng of the prepped library following the protocol for Agilent's SureSelect Human All Exon v5 + UTRs 75 MB kit. The purified capture products were amplified using the SureSelect Post-Capture Indexing forward and Index PCR reverse primers (Agilent) for 12 cycles. Libraries were sequenced at an average coverage of ~80X following Illumina's standard protocol in an Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 150 X 2 paired end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling is performed using Illumina's RTA version 2.7.3. Data was processed through an in-house bioinformatics pipeline and analyzed using Ingenuity (Qiagen) by the Center for Individualized Medicine (Mayo Clinic).
Ferrer A., Starosta R.T., Ranatunga W., Ungar D., Kozicz T., Klee E., Rust L.M., Wick M, & Morava E. (2020). Fetal glycosylation defect due to ALG3 and COG5 variants detected via amniocentesis: Complex glycosylation defect with embryonic lethal phenotype. Molecular genetics and metabolism, 131(4).
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