Qubit dsDNA HS Assay Kit

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Do not label the side of the tube as this could interfere with the sample read.

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Do not label the side of the tube as this could interfere with the sample read.

Publication protocol

GRO-seq assay
GRO-seq libraries were produced for A549, ARPE, HEK293T, HeLa, HepG2, hESC9, HUVEC, MRC5, NHA, T98G, SKOV3, THP-1 and U87 cells, to be integrated with our earlier data from HAEC, HUVEC, K562, LNCaP, Nalm6, REH, VCAP and other public GRO-seq samples (in AC16, H1-ESC, HCT116, IMR90, MCF7, Ramos, GM12004 and GM12750 cells). Cell lines were cultured following the ATCC guidelines and replicate samples were included to confirm reproducibility (see Supplementary Figure S1).

For the run-on assay, phosphate-buffered saline-washed cells were incubated in 10 ml of swelling buffer (10 mM Tris–HCl, 2 mM MgCl2, 3 mM CaCl2 and 2 U/ml SUPERase Inhibitor (Thermofisher, Carlsbad, CA, USA) RNAse inhibitor) for 5 min on ice. Cells were pelleted for 10 min at 400 × g and resuspended in 500 μl of swelling buffer supplemented with 10% glycerol. Subsequently, 500 μl of swelling buffer supplemented with 10% glycerol and 1% Igepal was added drop by drop to the cells under gentle vortexing. Nuclei were washed twice with lysis buffer (10 ml of swelling buffer supplemented with 0.5% Igepal and 10% glycerol), and once with 1 ml of freezing buffer (50 mM Tris–HCl pH 8.3, 40% glycerol, 5 mM MgCl2 and 0.1 mM ethylenediaminetetraacetic acid). Nuclei were counted, centrifuged at 900 × g for 6 min and suspended to a concentration of 5 million nuclei per 100 μl of freezing buffer, snap-frozen in LN2 and stored −80°C until run-on reactions. The nuclear run-on reaction buffer (NRO-RB; 496 mM KCl, 16.5 mM Tris–HCl, 8.25 mM MgCl2 and 1.65% Sarkosyl (Sigma-Aldrich, Steinheim, Germany) was pre-heated to 30°C. Then each ml of the NRO-RB was supplemented with 1.5 mM DTT, 750 mM adenosine triphosphate, 750 mM GTP, 4.5 mM CTP, 750 mM Br-UTP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and 33 ml of SUPERase Inhibitor (Thermofisher, Carlsbad, CA, USA). A total of 50 μl of the supplemented NRO-RB was added to 100 μl of nuclei samples, thoroughly mixed and incubated for 5 min at 30°C. GRO-seq libraries were subsequently prepared as previously described (17). Briefly, the run-on products were treated with DNAse I according to the manufacturer's instructions (TURBO DNA-free Kit, Thermofisher, Carlsbad, CA, USA), base hydrolysed (RNA fragmentation reagent, Thermofisher, Carlsbad, CA, USA), end-repaired and then immuno-purified using anti-Br-UTP beads (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Subsequently, a poly-A tailing reaction (PolyA polymerase, New England Biolabs, Ipswich, MA, USA) was performed according to manufacturer’s instructions, followed by circularization and re-linearization. The cDNA templates were polymerase chain reaction (PCR) amplified (Illumina barcoding) for 11–14 cycles and size selected to 180–300 bp length. The ready libraries were quantified (Qubit dsDNA HS Assay Kit on a Qubit fluorometer, Thermofisher, Carlsbad, CA, USA) and pooled for 50 bp single-end sequencing with Illumina Hi-Seq2000 (GeneCore, EMBL Heidelberg, Germany).

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