Illumina NovaSeq 6000 paired end sequencing; Dual role of EZH2 on megakaryocyte differentiation, ChIP-seq analysis at different stages of megakaryocyte maturation
Dual role of EZH2 on megakaryocyte differentiation, ChIP-seq analysis at different stages of megakaryocyte maturation
Cord blood samples were obtained after informed consent in agreement with our Institute Ethic Committee (Assistance Publique des Hôpitaux de Paris) and in accordance with the Declaration of Helsinki. They were processed and separated using immunomagnetic beads following manufacturer's protocol (Miltenyi Biotech). Isolated CD34+ cells were cultured in serum-free medium in presence of recombinant human Thrombopoietin (TPO) and Stem Cell Factor (SCF). EZH2 inhibition was achieved using GSK343 (Sigma-Aldrich,) chemical inhibitor at a concentration of 1µM added to culture medium at day 0 and each three subsequent days.  For the ChIP-seq, all the samples were cross-linked with a 1% formaldehyde solution for 15 min at RT. Cross-linking was stopped by addition of glycine to 125 mM final concentration. Cells were washed twice with PBS 1X and frozen at -80°C. Lysis of the cell membrane was performed (Hepes-KOH pH7.5 10mM, EGTA pH8 0.5 mM, EDTA pH8 10 mM and Triton-X 0.25%, protease inhibitor, Roche) followed by lysis of the nuclear membrane (Hepes-KOH pH7.5 10mM, EGTA pH8 1 mM, EDTA pH8 0.5 mM and NaCl 200mM, protease inhibitor, Roche). Samples were sonicated to generate DNA fragments below 500 bp. To remove background, samples were pre-cleared by adding Dynabeads protein A (Life Technologies, 10001D). Samples were then incubated overnight with following antibodies 5 g of H3K27me3 (CST #9733S), 5 g of H3K27Ac (Actif Motif #39133), 5 g of H3K4me3 (Diagenode #C1541003). For the immunoprecipitation Dynabeads protein A (Life technologies, 10001D) were added to each sample and incubated for 90 minutes at 4°C. Beads were then washed once with ChIP buffer (EDTA pH8 5 mM, NaCL 150 mM, Tris HCl pH7.5 25 mM, Triton-X 1%, Protease inhibitors, Roche) and twice using ChIP buffer and NaCl at a final concentration of 500mM. Immunocomplexes were eluted for 20 min at 65°C with 1% SDS and cross-linking was reversed by adjusting to 200 mM NaCl and incubating over/night at 65°C. DNA was purified using phenol/chloroform/alcohol and subjected to NGS sequencing. Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an 'A' base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific) using the Bravo Platform (Agilent), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter) and amplified by PCR for 10 cycles more.