title: GSM5048186: H3K27me3 young rep3; Mus musculus; ChIP-Seq
library_construction_protocol: Skin pieces were incubated in 0.8% trypsin in PBS for 60 minutes at 37°C. After incubation, the epidermis was manually detached from the dermis, single cell epidermal suspensions where generated by pippeting and collected by centrifugation. Cells were labeled with antibodies against Cd34 and alpha6 integrin, after which Cd34 alpha6 integrin-double positive cells were isolated by flow cytometry. H3K4me3, H3K27me3 and Pol2 ChIP-Seq experiments were performed using Diagenode's ChIPmentation Kit for Histones (C01011010). Briefly, freshly purified HFSCs were crosslinked with 0.25% methanol free formaldehyde (ThermoFisher 28906) for 10 min at room temperature. Fixation was terminated by adding 0.125 M Glycine in PBS for 5 min at room temperature. Cells were subsequently washed with PBS after which cells were pelleted by centrifugation at 5000 RPM for 10 minutes at 4C. Cell pellets were lysed through 10 strokes of Dounce homogenization to aid nuclear release. Lysates were sonicated using a Covaris M20 sonicator (4-9 C temperature range; 15% duty factor 75% peak power) for 10 min. After sonication, samples were subjected to centrifugation at 21000xg for 5 min at 4 C and the supernatant were collected as the chromatin fraction. DNA concentration was measured using Qubit DS High-sensitivity kit (Invitrogen) and sample concentrations were adjusted to be equal. 10% of chromatin was collected as input and 2 µl was analyzed using Agilent 2200 TapeStation to ensure optimal shearing (300-600 bp fragments) and the rest was subjected to immunoprecipitation. For this, sheared chromatin, DiaMag Protein A-coated beads and 3 µg of antibody (H3K27me3, Active motif #39155; H3K4me3, Cell Signaling #9727; or Pol2, Cell Signaling #14958) were combined to a ChIP reaction mix and incubated overnight at 4 C. Beads were collected using a magnet, washed, resuspended in tagmentation buffer containing 1 ul of tagementation enzyme and incubated for 10 min at 37C. After several washes, bound complexes were stripped of the beads, end repaired and cross-linking was reversed. For ChIPmentation libraries, the optimal cycle number for library amplification was determined by qPCR (12-14 cycles) and library prepared for sequencing by PCR using barcoded primer sets.