Qubit dsDNA HS Assay Kit

Protocol tips

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

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

Publication protocol

ChIP library construction
ChIPed DNA was size separated using 8% polyacrylamide gel electrophoresis (PAGE). The 200–500-bp DNA fractions were excised from the gel and were eluted from the gel slice overnight at 4 °C in elution buffer. Elution buffer consisted of a 5 to 1 ratio of LoTE buffer (3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA) and 7.5 M ammonium acetate. DNA was purified using a Spin-X Filter Tube (Fisher Scientific, UK), and ethanol precipitated.

Library construction was carried out on the Bravo liquid handling platform using VWorks Automation Control Software (Agilent Automation). Samples were first subjected to end repair using T4 polynucleotide kinase (NEB), T4 DNA polymerase (NEB) and Klenow DNA polymerase (NEB) at room temperature for half an hour. DNA was purified using PEG-Sera Mag Speedbeads (Fisher) with 13.87% final polyethylene glycol (PEG) concentration. A-tailing was then performed using Klenow exo minus (NEB) at 37 °C for 30 min. Products were purified as before. Illumina short sequencing adaptors were ligated to A-tailed product using T4 DNA ligase (NEB) at room temperature overnight. To remove adaptor dimers and library fragments below 200 bp, products were purified twice using PEG-Sera Mag Speedbeads with 8.89% and 10.91% final PEG concentrations. Adaptor ligated libraries were PCR amplified and barcoded using custom indexing primers, Illumina PCR primer 1.0 and 0.5 U of Phusion Hot Start II (Fisher). The initial denaturation step at 98 °C for 30 s was followed by 13 cycles of 15 s at 98 °C, 30 s at 65 °C and 30 s at 72 °C, and a final step at 72 °C for 5 min. Amplified libraries were purified using PEG-Sera Mag Speedbeads with 9.19% final PEG concentration. Libraries were quantified using a Qubit HS DNA assay (Invitrogen) and equal molar amounts were pooled. Each pool was quantified for sequencing using the Kapa SYBR Fast Complete Universal qPCR kit (Kapa Biosystems).

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