ChIP-Atlas: SRX1924142

Antigen

Cell type Class: Blood
Cell type: Hematopoietic Stem Cells
MeSH Description: Progenitor cells from which all blood cells derived. They are found primarily in the bone marrow and also in small numbers in the peripheral blood.

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: HSCs purified as indicated above were sorted and crosslinked with 1% formaldehyde at room temperature (RT) for 10 min, and the reaction was stopped by 0.125M glycine at RT for 5 min. Cross-linked cells were lysed and sonicated to 200-500 bp fragments (Bioruptor, Diagenode). ChIP-qualified antibodies (H3K27me3 Millipore 07-449) were added to the sonicated chromatin and incubated at 4°C overnight. Following this, 10 μl of protein A magnetic beads (Dynal, Invitrogen) previously washed in RIPA buffer were added and incubated for an additional 2 hours at 4°C. The bead: protein complexes were washed three times with RIPA buffer and twice with TE buffer. Following transfer into new 1.5 ml collection tube, genomic DNA was eluted for 2 hours at 68°C in 100 µl Complete Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 µg/ml proteinase K), and combined with a second elution of 100 µl Elution Buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl) for 10 min at 68°C. ChIPed DNA was purified by MinElute Purification Kit (Qiagen) and eluted in 12 µl elution buffer. ChIP-seq library is constructed using Rubicon Genomics ThruPLEX DNA-seq Kit following the manufacture's protocol.