GSM7286724: 2xMinikirikou-BLS_12-7F5; Gallus gallus; MNase-Seq
We cross-linked 30 x 106 exponential growing cells by incubation for 5 minutes with 1% freshly prepared formaldehyde (Thermo Fisher Scientific, #28908) at room temperature. Fixation was stopped by adding 0.125 M glycine-PBS for five minutes at room temperature. After three ice cold PBS washing, nuclei were extracted in lysis buffer (10mM Tris-HCl, pH7.5, 10mM NaCl, 3mM MgCl2, 0.2% triton X-100, 0.5mM EGTA, 1mM DTT, 1x protease inhibitor cocktail (Sigma, #P8340)) for 5min on ice, 15 centrifuged and resuspended in digestion buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 1x protease inhibitor cocktail (Sigma, #P8340)). Micrococcal Nuclease (MNase; Thermo Fisher Scientific, #EN0181) digestions were performed for 15 minutes at 37°C, with 160 U/mL of MNase. The reaction was stopped by adding 0.1 volume of stop buffer (200 mM EDTA pH 8.0, 40 mM EGTA pH 8.0). Chromatin was then sonicated during 30 sec at 7°C in a buffer adjusted to a final SDS concentration of 0.1% with a Covaris sonicator using 75W intensity, 200 cycles per burst, 5% of duty cycle. For nucleosome coverage analyses, DNA molecules were recovered after RNAse A and proteinase K digestion and phenol-chloroform extraction and ethanol precipitation. Sequencing libraries were prepared using NEBNext Ultra II DNA library prep Kit for Illumina (NEB #E7645S) according to the manufacturer's instructions for MNase, ChIp and input libraries. Samples were not subjected to size selection, but only cleaned-up for adaptor-ligated DNA using SPRISelect Reagent kit (Beckman coulter #B23317), to ensure a MNase digested small fragment overall recovery. Libraries were prepared with a starting DNA input of 10 ng. We used a 10 fold diluted adaptor for the adaptor ligation. The library amplification was performed using NEB-Next Multiplex Oligos for Illumina (NEB #E7710S) with different NEB-Next index primers and the NEB-Next Universal PCR Primer (NEB #E6861A) with eight PCR cycles.