ChIP-Atlas: SRX4521416

Antigen

Cell type Class: Blood
Cell type: Bone Marrow Cells
MeSH Description: Cells contained in the bone marrow including fat cells (see ADIPOCYTES); STROMAL CELLS; MEGAKARYOCYTES; and the immediate precursors of most blood cells.

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: Cells were lysed in non-ionic detergents and protease inhibitor cocktail. Cells were digested by Micrococcal nuclease (MNase) at room temperature for 5 minutes and 0.25mM EDTA was used to stop the reaction. H3K27me3 antibody was incubated with anti-IgA magnetic beads for 2 hours. Digested chromatin was incubated with magnetic beads alone for 1.5 hours. Digested chromatin was separated from beads and incubated with antibody-bead complex overnight in IP buffer. IPs were washed twice and eluted for 1.5 hours. Histones were digested by Protease for 30min and DNA fragments were purified using Sera Mag magnetic beads in 30% PEG. The PCR program included an initial denaturation step at 98C for 30 sec, followed by 8 cycles at 98C for 15 sec, 65C for 30 sec, and 72C for 30 sec, and ending with a final step at 72C for 5 min. We performed native chromatin immunoprecipitation (ChIP) as described previously (Lorzadeh et al., 2016) using a validated anti-H3K27me3 antibody (cat #pAB-195-050,Diagenode). Libraries were constructed using an Agilent Bravo automated liquid handling platform. Illumina sequencing libraries were generated by end repair, 3' A-addition, and Illumina sequencing adaptor ligation.