GSM1253297: ESC; Mus musculus; OTHER
One million cells were used to construct RED-seq libraries. Cells were washed, pelleted, and resuspended in Swelling buffer (10mM Tris pH8.0, 85mM KCl, 0.5% NP-40, 10mM MgCl2) with 100 units of Sau96I (NEB) and incubated in a thermomixer (Eppendroff) at 37°C for 1 hour, shaking at 900 rpm. Digestion was terminated by adding 40μl of 10% SDS and 20μl of 0.5M 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 according to Illumina's instructions., 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 250μl with streptavidin binding buffer (20mM HEPES pH 7.6, 500mM NaCl, 1mM EDTA, 0.02% NP-40) and incubated with 30μl 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).
GEO Accession
GSM1253297