GSM2417333: MNase-seq_Control_rep1,rep2; Mus musculus; MNase-Seq
Biological replicates were obtained from two independent transfection experiments for each shRNA vector. Briefly, ~5 million cells were crosslinked with 1% formaldehyde for 20 min followed by quenching for 5 min with the addition of glycine to a final concentration of 0.125 M. After washing with PBS buffer, cells were collected and lysed in Cell Lysis buffer (5 mM Tris pH8.0, 85 mM KCl, 0.5% NP40 ) with proteinase inhibitors (10 µl/mL Phenylmethylsulfonyl fluoride (PMSF), 1 µl/mL Leupeptin and 1 µl/mL Pepstatin). Nuclei were gathered by centrifugation (5000 rpm for 2 min) and were treated with 10 Kunitz Units/106 cells of micrococcal nuclease (NEB, #M0247S) for 5 min at 37°C. The reaction was stopped with the addition of 60 µl 50 mM EDTA, 25 µl 5 M NaCl, 15 µl 20% NP-40 and incubated on a rotator for 1h at room temperature to release soluble nucleosomes. Samples were centrifuged for 5 min at 10,000 g and supernatant was transferred to a new tube. Samples were reverse-crosslinked by incubating overnight at 65°C with 0.5% SDS and proteinase K. Following phenol-chloroform extraction and ethanol precipitation, DNA was incubated at 37°C for 4h with RNAse (Sigma). All the samples were run in a 2% agarose gel and fragments <200 bp were extracted and purified using the NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel) according to the manufacturer’s instructions. Purified DNA (500 ng) was used for library preparation as described previously (Tessarz, et al). The only difference was the PCR amplification step where we used the same conditions as mentioned in Henikoff et al but with only three amplification cycles.
GEO Accession
GSM2417333