Sau96I NEB#R0165

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion).	Digestion was terminated by adding 40 μl of 10% SDS and 20 μl of 0.5 M EDTA and the chromatin was treated with proteinase K (Ambion) overnight at 55°C. Digested DNA was purified using phenol/chloroform/isoamyl alcohol extractions and precipitated at -80°C for 1 hour. Digested DNA samples were end-repaired and A-tailed as described [63], and ligated with biotinylated and barcoded adaptors.

Protocol tips
Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion).
Downstream tips
Digestion was terminated by adding 40 μl of 10% SDS and 20 μl of 0.5 M EDTA and the chromatin was treated with proteinase K (Ambion) overnight at 55°C. Digested DNA was purified using phenol/chloroform/isoamyl alcohol extractions and precipitated at -80°C for 1 hour. Digested DNA samples were end-repaired and A-tailed as described [63], and ligated with biotinylated and barcoded adaptors.

Publication protocol

"One million cells were used to construct RED-seq libraries. Cells were washed, pelleted, and resuspended in swelling buffer (10 mM Tris pH8.0, 85 mM KCl, 0.5% NP-40, 10 mM MgCl2) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. (For testing whether two REs might increase coverage, in one experiment 100 units of Sau96I and 50 units of DdeI were used in digestion). Digestion was terminated by adding 40 μl of 10% SDS and 20 μl of 0.5 M EDTA and the chromatin was treated with proteinase K (Ambion) overnight at 55°C. Digested DNA was purified using phenol/chloroform/isoamyl alcohol extractions and precipitated at -80°C for 1 hour. Digested DNA samples were end-repaired and A-tailed as described [63], and ligated with biotinylated and barcoded adaptors. DNA was purified using Zymo Research DNA clean and concentrate columns following each enzyme reaction. The biotin-adaptor ligated DNA was sonicated in a Covaris sonicator (S220) to generate DNA peak fragments of 200 bp, on average. The sonicated DNA samples were then end-repaired, A-tailed, and ligated with non-biotinylated adaptors. The ligation samples were loaded on 2% agarose gel and DNA was purified within a size range of roughly 200-350 bp in length. Gel-purified DNA was diluted to 250 μl with streptavidin binding buffer (20 mM HEPES pH 7.6, 500 mM NaCl, 1 mM EDTA, 0.02% NP-40) and incubated with 30 μl of pre-washed streptavidin magnetic beads (NEB) at room temperature for 1 hour. After magnetic separation, the supernatants were removed, and the beads were washed additional three times with streptavidin binding buffer. DNA was eluted from streptavidin magnetic beads by adding 20 μl of 0.1X TE and heating at 60°C for 3 minutes. The elution was repeated three times. The adaptor-ligated material was then PCR amplified with Phusion polymerase (NEB) using 16 cycles of PCR and its concentration was determined using a NanoDrop (Thermo). The integrity of each library was confirmed by sequencing 10-20 individual fragments per library. Libraries with different barcodes were pooled together and single-end sequencing (50 bp) was performed on an Illumina HiSeq2000 at the UMass Medical School deep sequencing core facility.

For most RED-seq libraries (GFP, Oct4, Ep400 and Hira KD), we added one further modification in which the sequence of the biotinylated adapters and the second, non-biotinylated, adapters were modified such that after PCR amplification of the libraries, only the end that was ligated to the biotinylated adapter would be sequenced in a single-end sequencing run (Additional file 3). Although this alteration makes the data analysis slightly simpler, the two methods provide essentially identical results."

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