title: GSM4261055: Ctrl H3K3me3; Mus musculus; ChIP-Seq
library_construction_protocol: Aliquots of 100,000 Th17 cells were thawed on ice for 5 min and immediately resuspended in 20uL of EZ Nuclei Isolation Buffer (Sigma-Aldrich), and incubated for 5 min on ice. Samples were digested using 2 Units/uL MNase in 20uL MNase Digestion Buffer (NEB) for 5 minutes at 37oC. Reactions were stopped by the addition of 10mM EDTA and 0.1% Trition/0.1% Deoxycholate (final concentration). Isolated chromatin was incubated on ice for 15 min, followed by vortexing on medium setting for 30 sec. The volume was adjusted to 200uL with Complete IP Buffer (20mM Tris-HCL pH 8.0, 2mM EDTA, 150mM NaCl, 0.1% Triton X- 100, 10mM Sodium Butyrate, 1x Protease Inhibitor Cocktail, 1mM PMSF), and incubated for 1 h at 4oC on a gentle rocking platform. 10% of the total chromatin was removed to assess digestion efficiency and to use as an input control. Chromatin for immunoprecipitation was pre-cleared using Protein A Dynabeads (Invitrogen) for 1–4 hours at 4oC and subjected to IP overnight at 4oC (0.03μg H3K4me3 Abcam ab8580; 0.2μg H3K4me1 Abcam ab8895). Bead-chromatin complexes were washed using low-salt wash buffer (0.1% SDS, 1% Trition X-100, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl, 1x Protease Inhibitor Cocktail, 10mM Sodium Butyrate), high-salt wash buffer (0.1% SDS, 1% Trition X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl, 1x Protease Inhibitor Cocktail, 10mM Sodium Butyrate). Chromatin was eluted from the beads using elution buffer (1% SDS, 100mM NaHCO3) by shaking for 1 h at 65oC. DNA was then purified by phenol-chloroform extraction using Maxtract tubes (Qiagen) and then ethanol precipitated overnight. Immunoprecipitated DNA was prepared for sequencing on the Illumina platform using the NEBNext ChIP-Seq Library Prep Master Mix Set using modified Illumina TruSeq adapters.
attributes.tag: GEO Accession
attributes.value: GSM4261055