GSM4551825: Adult_CTX_SSTcre_Dnmt3aKO_Sun1IP_H3K27ac_rep2; Mus musculus; ChIP-Seq
INTACT-purified nuclei were crosslinked in 1% formaldehyde, followed by the addition of 0.125M glycine to stop crosslinking activity. The nuclei were washed with cold 1XPBS, then lysed once with L1 buffer (50mM HEPES pH7.5, 140mM NaCl, 1mM EDTA, 1mM EGTA, 0.25% Triton X-100, 0.5% NP40, 10% Glycerol, protease inhibitors, sodium butyrate), followed by an incubation in L2 buffer (10mM Tris-HCl pH8.0, 200mM NaCl, protease inhibitors, sodium butyrate). The nuclei were resuspended in L3 buffer (1M Tris-HCl pH8.0, 5M NaCl, 0.5 M EDTA, 0.5 M EGTA, 10% Na-Deoxycholate, 20% N-lauroylsarcosine, protease inhibitors, sodium butyrate) and sonicated with a Bioruptor (Diagenode) for 40 cycles of 30 seconds “on” and 45 seconds “off.” Sonicated chromatin were centrifuged at 13,000 rpm for 10 minutes at 4°C to remove insoluble pellet, and then pre-cleared by incubating with Protein A Dynabeads (Thermofisher) for 2 hours at 4°C. Pre-cleared chromatin were incubated with Dynabeads conjugated to H3K27ac (Abcam ab4729) or H3K4me1 (Abcam ab8895) antibodies overnight at 4°C. Beads were washed twice each with Low Salt buffer (0.1% SDS, 1% Triton X-100, 20mM Tris HCl pH8.0, 150mM NaCl, 2mM EDTA), High Salt buffer (0.1% SDS, 1% Triton X-100, 20mM Tris HCl pH8.0, 500mM NaCl, 2mM EDTA) and LiCl buffer (250mM LiCl, 1% NP40, 1mM EDTA, 10mM TrisHCl pH8.0, 1% Sodium Deoxycholate) at 4°C, and washed once with 1XTE buffer at room temperature. Immunoprecipitated chromatin were first eluted off the beads with 1XTE+1%SDS at 65°C with shaking for 30 minutes, and then were de-crosslinked overnight at 65°C. RNAseA was added to each sample, followed by an incubation at room temperature for 30 minutes. The samples were incubated with proteinase K for two to three hours at 55°C with shaking. Immunoprecipitated DNA were purified using phenol-chloroform-isoamyl alcohol followed by Qiagen MinElute columns. Libraries were constructed by using Ovation Ultralow Library Systems (Nugen) following manufacturer instructions.
GEO Accession
GSM4551825