Quant it high sensitivity assay

Manufactured by Thermo Fisher Scientific

The Quant-iT High-Sensitivity assay is a fluorescence-based method for quantifying small amounts of DNA, RNA, or protein samples. It provides sensitive and accurate measurements across a wide linear dynamic range.

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Lab products found in correlation

Bcl2fastq software, by Illumina (2 mentions) Lpnpi, by Thermo Fisher Scientific (2 mentions) Lpnpi, by New England Biolabs (2 mentions) Agarose gel cassettes, by Sage Science (2 mentions) Thruplex dna seq 96d kit, by Takara Bio (2 mentions) Hiseq 2500 system, by Illumina (2 mentions) Mspji, by New England Biolabs (1 mentions) Pippinht system, by Sage Science (1 mentions)

2 protocols using quant it high sensitivity assay

MeD-seq Analysis of iPSC DNA

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DNA from iPSC samples collected at passage 12 were used for MeD‐seq analysis. LpnPI and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol. Reactions contained 50 ng in a 10‐μL volume and digestion took place overnight in the absence of enzyme activators. Digests of genomic DNA with LpnPI resulted in snippets of 32 bp around the fully methylated recognition site that contains CpG. The DNA concentration was determined by the Quant‐iT High‐Sensitivity assay (Life Technologies; Q33120) and 50 ng ds DNA was prepared using the ThruPlex DNA‐seq 96D kit (Rubicon Genomics cat#R400407). Twenty microliters of amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science). Stem‐loop adapters were blunt‐end ligated to repaired input DNA and amplified (4 + 10 cycles) to include dual‐indexed barcodes using a high fidelity polymerase to yield an indexed Illumina NGS library. Multiplexed samples were sequenced on Illumina HiSeq2500 systems for single read of 50 base pairs according to the manufacturer's instructions. Dual‐indexed samples were demultiplexed using the bcl2fastq software (Illumina).

Kenter A.T., Rentmeester E., van Riet J., Boers R., Boers J., Ghazvini M., Xavier V.J., van Leenders G.J., Verhagen P.C., van Til M.E., Eussen B., Losekoot M., de Klein A., Peters D.J., van IJcken W.F., van de Werken H.J., Zietse R., Hoorn E.J., Jansen G, & Gribnau J.H. (2020). Cystic renal‐epithelial derived induced pluripotent stem cells from polycystic kidney disease patients. Stem Cells Translational Medicine, 9(4), 478-490.

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Methylation-Sensitive Fragmentation and NGS Library Prep

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LpnPI (New England Biolabs) digestions were carried out on DNA samples according to the manufacturer's protocol. Reactions contained 50 ng in a 10 μL volume and digestion took place overnight in the absence of enzyme activators. Digests of genomic DNA with LpnPI resulted in snippets of 32 bp around the fully-methylated recognition site that contains CpG. The DNA concentration was determined by the Quant-iT™ High-Sensitivity assay (Life Technologies; Q33120) and 50 ng ds DNA was prepared using the ThruPlex DNA-seq 96D kit (Rubicon Genomics cat#R400407). Stem-loop adapters were blunt end ligated to repaired input DNA and amplified (4 +10 cycles) to include dual indexed barcodes using a high fidelity polymerase to yield an indexed Illumina NGS library. Twenty microliters of amplified end product was purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science). Multiplexed samples were sequenced on Illumina HiSeq2500 systems for single read of 50 base pairs according to manufacturer's instructions. Dual indexed samples were demultiplexed using bcl2fastq software (Illumina).

Soochit W., Sleutels F., Stik G., Bartkuhn M., Basu S., Hernandez S.C., Merzouk S., Vidal E., Boers R., Boers J., van der Reijden M., Geverts B., van Cappellen W.A., van den Hout M., Ozgur Z., van IJcken W.F.J., Gribnau J., Renkawitz R., Graf T., Houtsmuller A., Grosveld F., Stadhouders R, & Galjart N. (2021). CTCF chromatin residence time controls three-dimensional genome organization, gene expression and DNA methylation in pluripotent cells. Nature cell biology, 23(8).

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