1.0x104 HSCs were sorted into PBS+10% FBS, and then crosslinked at room temperature for 8 minutes using 1% paraformaldehyde (VWR #J531).  Chromatin was sheared using a Covaris E220 Focused-ultrasonicator until the DNA fragments were between 200 – 700 base pairs.  Chromatin samples were incubated overnight at 4°C with either the H3K27me3 antibody (Diagenode # pAb-195-050).  Following overnight incubation, protein G dynabeads (Life Technologies #130-099-508) were added for 2-hours at 4°C.  The beads were washed, and tagmented for 10-minutes at 37°C using the Nextera DNA Library Preperation Kit (Illumina #FC-121-1030).  After additional washes, an overnight decrosslinking was performed immunoprecipiated DNA was isolated using Ampure XP beads. 2xKapa HiFi HotStart Ready Mix (Kapa #KB KK2601), along with Nextera custom primers, was used for library amplification of the ChIP DNA(12 cycles of PCR as determined by qPCR).  Libraries were again purified with Ampure XP beads and run on an Illumina Hiseq 3000 (PE2X150).
GEO Accession
GSM3405446