ChIP-Atlas: SRX1092619

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.

source_name: Multipotent progenitors
strain: C57BL/6J
material source: bone marrow from 8-week old C57BL/6J mice
sorting: lin-, Sca1+, c-Kit+, Flk2+, CD34+
cell type: Multipotent progenitors
chip antibody: anti-H3K4me3 (Cell Signaling, catalog# 9751S, 11/2014, lot# 8)

library_strategy: ChIP-Seq
library_source: GENOMIC
library_selection: ChIP
library_construction_protocol: mESCs (500 or 1000) or sorted HSC subtypes were mixed with ~5x108 DH5a E.coli and fixed in 1% formaldehyde for 10 min at room temperature. After fixation and washes, the mixtures were sonicated with a 1/16-inch probe for 15 min at 3 watts using a tip sonicator (Misonix sonicator 3000). The lysates were incubated with 2 microliter H3K27me3 antibody (Millipore 07-449) or 1 microliter H3K4me3 antibody (Cell Signaling 9751S) overnight at 4oC. To block any non-specific chromatin absorption, 60 microliter protein G magnetic beads (Life Technologies 10004D) or 10 microliter M-280 streptavidin beads (Life Technologies 11206D) were pre-incubated with ~5x108 fixed and sonicated E.coli lysate or the same E.coli lysate plus 5 ng carrier biotin-DNA, respectively, at 4oC overnight. These treated beads were combined and used to ChIP H3K27me3 or H3K4me3 in the sonicated E.coli+mESC or E.coli+HSC lysates for 4 h at 4oC. The beads were then recovered using a magnetic stand. All washing, de-crosslinking, and library building steps were performed following Illumina True-Seq protocol with 12 PCR amplification cycles. After end repair, A-tailing, and adapter ligation steps, 0.25 micro-M blocker oligo was added during final library amplification.