title: GSM3070320: Rybp_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Basal layer epidermal progenitors were isolated from Krt14-RFP and Krt10-H2B-GFP newborn mice. Briefly, P0 back skins were collected and incubated for 4-6 hours in 1.26U/mL dispase (Invitrogen) at 4ºC. The epidermis was gently peeled from the underlying dermis, dissociated by 0.25% Trypsin with 2.21mM EDTA (Corning Cellgro; Manassas, VA, USA) and washed twice with 1x PBS. Cells were stained with 1:200 Sca1-APC ((Biolegend; San Diego, CA, USA) for 30 minutes on ice and washed twice with 1x HBSS prior to cell sorting. Epidermal progenitors were sorted as Sca1(+), Krt14-RFP(high), and Krt10-H2B-GFP(-). A total of 0.4 x 10^6 cells was used for each histone mark per ChIP, and a total of 6 x 10^6 cells was used for each Polycomb subunit per ChIP.  Prior to cell sorting, cells were stained for viability using Zombie violet (Biolegend; San Diego, CA), then cross-linked using fresh solution with a final concentration of 1% formaldehyde (Thermo Fisher Scientific; Rockford, IL) for 10 minutes at room temperature. Crosslinking was stopped by the addition of Glycine (final concentration 125mM) for 5 minutes of incubation at room temperature, followed by two washes with 1x PBS. Cells were incubated in lysis buffer 1 (50mM HEPES pH=7.5, 140mM Nacl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, protease inhibitor cocktail (Roche)) for 10 min on ice, then incubated for 10 min with lysis buffer 2 (10mM Tris-HCl pH=7.5, 200mM NaCl, 1mM EDTA, 0.5mM EGTA). Before ChIP, cells were resuspended in lysis buffer 3 (10mM Tris-HCl pH=8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-deoxycholate, 0.5% N-laurylsarcosine, 1% Triton X-100) and sonicated using a Bioruptor Sonicator (Diagenode, UCD-200) according to a 25x regimen of 30 seconds of sonication followed by 90 seconds of rest at 2.7ºC.  Chromatin was incubated overnight at 4ºC with antibodies as indicated in Table S5.  Dynal protein G magnetic beads (Invitrogen) were added the next day and incubated for 4 hours. The beads were sequentially washed with low salt, high salt, LiCl, and Tris-EDTA buffers for 10 minutes each at 4ºC. Bound chromatin was eluted and crosslinking was reversed by overnight incubation at 65ºC, followed by RNase A (Sigma-Aldrich) and proteinase K (Roche Diagnostics) treatments.  Samples were purified using ChIP DNA Clean and Concentrator kit (Zymo Research; Irvine, CA). For high-throughput ChIP sequencing, libraries were constructed from 3ng of Purified DNA using the DNA SMART ChIP-Seq Kit (Clontech; Palo Alto, CA, USA) according to the manufacturer's instructions. Constructed ChIP-seq libraries were sequenced on the Illumina HiSeq 4000 platform
attributes.tag: GEO Accession
attributes.value: GSM3070320