Ficoll hypaque gradient separation

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor buffycoats by Ficoll–Hypaque gradient separation (GE-Healthcare). NK isolation from PBMCs was performed by negative selection using the NK cell Isolation Kit (Miltenyi Biotec GmbH, GE) according to manufacturer’s instructions. Purity of enriched polyclonal NK cells was measured as percentage of CD56⁺ CD3⁻ population by flow cytometry and confirmed to be >95%. Purified polyclonal NK cells were maintained in RPMI 1640 supplemented with 10% FBS and 100 U/ml of r-IL-2 (R&D System Inc., MN, USA).

Blasts cells were purified by the Ficoll-Hypaque gradient separation method (GE Healthcare). RNA was obtained by using TRIzol (Life Technologies) according to the manufacturer’s recommendations. Isolation of genomic DNA has complied the FlexiGene DNA protocol (Qiagen).
ChIP extracts preparation has been carried out following IHEC procedures and as reported in [47 (link), 48 (link), 53 (link), 54 (link)].

Buffy coats
from anonymous donors were obtained from the Karolinska University
Hospital, Stockholm (see above). NK cells were obtained through negative
selection using the NK cell isolation kit (Miltenyi Biotec Ltd.).
In brief, peripheral blood mononuclear cells (PBMCs) were isolated
by Ficoll–Hypaque gradient separation (GE-Healthcare), followed
by incubation with biotinylated antibody cocktail (Miltenyi Biotec
Ltd.) at 4 °C. Antibiotin beads were then added for a further
15–20 min, and the cells were washed and added to magnetic
columns. The purity of the enriched polyclonal NK cells was measured
as percentage of the CD56⁺ (BioLegend Cat. No. 317343)
CD3^– (BioLegend Cat. No. 362504) population using
a CyAN ADP LX 9-color flow cytometer (Beckman Coulter). Data were
analyzed by using FlowJo software.