Human cot 1

Manufactured by Thermo Fisher Scientific

Sourced in United States

Human Cot-1 is a laboratory product that serves as a highly repetitive DNA sequence. It is commonly used in molecular biology and genomics research to help identify and separate unique sequences from highly repetitive sequences in the human genome.

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Lab products found in correlation

Salmon sperm dna, by Thermo Fisher Scientific (2 mentions) Hybridization buffer, by Agilent Technologies (2 mentions) Blocking agent, by Agilent Technologies (2 mentions) Hiseq 2500 system, by Illumina (1 mentions) Kapa library quantification kit, by Illumina (1 mentions) Herculase 2 fusion enzyme kit, by Agilent Technologies (1 mentions)

Spelling variants of this product

Human Cot-1 DNA (49 citations) Human COT-1 DNA/mouse COT-1 DNA (2 citations)

3 protocols using human cot 1

Targeted Enrichment of Barcoded Libraries

Cited 2 times

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For targeted enrichment, 700–1200 ng of each barcoded library was mixed with 2× hybridization buffer (Agilent, Santa Clara, CA, USA), 10× blocking agent (Agilent), blocking oligonucleotides (Maricic et al., 2010 (link)), human Cot1 (Invitrogen, Carlsbad, CA, USA) and salmon sperm DNA (Invitrogen) (Maricic et al., 2010 (link); Koumbaris et al., 2016 (link)). Immunoprecipitated libraries were incubated with the biotinylated capture probes for 48 hours at 66 °C and were eluted by heating, as previously described (Tsangaras et al., 2014 (link)). Enriched samples were amplified using outwardbound adaptor primers (Tsangaras et al., 2014 (link)) and were quantified using the KAPA Library Quantification KitIllumina (KAPA Biosystems, Boston, MA, USA). The enriched barcoded libraries were then pooled equimolarly and were subjected to pairedend sequencing on an Illumina HiSeq 2500 system (Illumina, San Diego, CA, USA).

KERAVNOU A., IOANNIDES M., TSANGARAS K., LOIZIDES C., HADJIDANIEL M.D., PAPAGEORGIOU E.A., KYRIAKOU S., ANTONIOU P., MINA P., ACHILLEOS A., NEOFYTOU M., KYPRI E., SISMANI C., KOUMBARIS G, & PATSALIS P.C. (2016). Whole-genome fetal and maternal DNA methylation analysis using MeDIP-NGS for the identification of differentially methylated regions. Genetics Research, 98, e15.

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Dual-Color FISH Probe for Species Identification

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A two-color species-differentiation fluorescence in situ hybridization (FISH) probe was generated by nick translation of species-specific repeated DNA sequences: human Cot-1 (Invitrogen) was labeled with SpectrumGreen, and porcine ID Block-It Cot-1 (Empire Genomics) was labeled with SpectrumOrange. The mixture was hybridized to control specimens from both species to confirm specific hybridization. Tissue slides were fixed in methanol/acetic acid fixative (3:1) for 60 min. The slides were dehydrated in graded ethanol (70%, 90%, 100%) at 25°C for 2 min each and air dried. The probe (8–10 µL) was added to the slide and a cover slip was placed. The slides and probe were co-denatured in a HYBrite at 73°C for 2 min and then hybridized at 37°C overnight in a humidified chamber. The slides were washed in 0.4 × SSC/ 0.3% NonidetP-40 (Igepal, CA 630) at 72°C for 2 min and 2 × SSC/0.1% NonidetP-4 at 25°C for 1 min. DAPI II is applied and the slides are analyzed by fluorescent microscopy.

Eslam R.B., Croce K., Mangione F.M., Musmann R., Leopold J.A., Mitchell R.N, & Waxman A.B. (2017). Persistence and proliferation of human mesenchymal stromal cells in the right ventricular myocardium after intracoronary injection in a large animal model of pulmonary hypertension. Cytotherapy, 19(6), 668-679.

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Target Capture Probe Enrichment Protocol

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Target capture probes were used to enrich libraries using in-solution Hybridization [16 ,28 ]. Each barcoded library was mixed with 2x hybridization buffer (Agilent Technologies), 10x blocking agent (Agilent Technologies), blocking oligonucleotides, human Cot-1 (Invitrogen, Carlsbad, CA, USA) and salmon sperm DNA (Invitrogen) [16 ,25 (link),28 ,29 ]. The libraries were then incubated with the biotinylated capture probes for 48 hours at 66°C and were eluted by heating [16 ,27 ]. Enriched fetal-specific regions were amplified for 12 cycles using Herculase II Fusion Enzyme kit (Agilent Technologies) and outward bound adaptor primers [27 ]. Following quantification with the KAPA Library Quantification Kit Illumina (KAPA Biosystems, Boston, MA, USA) the amplified post-captured libraries were sequenced on a HiSeq 2500 sequencing system platform (Illumina, San Diego, USA).

Keravnou A., Ioannides M., Loizides C., Tsangaras K., Achilleos A., Mina P., Kypri E., Hadjidaniel M.D., Neofytou M., Kyriacou S., Sismani C., Koumbaris G, & Patsalis P.C. (2018). MeDIP combined with in-solution targeted enrichment followed by NGS: Inter-individual methylation variability of fetal-specific biomarkers and their implementation in a proof of concept study for NIPT. PLoS ONE, 13(6), e0199010.

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