GSM6943805: 4540_MFL_KIThi(100)CD71hi; Mus musculus; ChIP-Seq
Cells were crosslinked in 1% formaldehyde prior to sorting in PBS/2mM EDTA at room temperature for 10 minutes. Reaction was quenched with glycine. Cell pellets were washed in PBS, resuspended in 1 ml cell lysis buffer (Methods) and incubated on ice for 20 minutes. Nuclei were pelleted and resuspended in 1 ml nuclear lysis buffer, incubated on ice for 20 minutes and sonicated for 45 minutes at 4 degrees Celsius. After sonication, samples were spun at 21130×g for 5 minutes at 4C. Supernatant (~ 1 ml) was diluted using 4 ml IP Dilution Buffer (Methods). Samples were precleared using 50 ug of isotope-matched IgG and 50 ul A/G agarose beads. IP reactions were set up by adding A/G beads pre-bound with antibody of interest and rotated overnight at 4 degrees Celsius. On the second day, beads were washed and eluted twice with 100 ul Elution Buffer. After reverse crosslinking, DNA was purified using the QIAquick PCR Purification kit per the manufacturer's instructions. ChIP-seq libraries were prepared using Illumina's TruSeq ChIP sample preparation kit (Illumina, Cat#IP-202-1012) according to manufacturer's protocol.