An encapsulated lymphatic organ through which venous blood filters.
Attributes by original data submitter
Sample
source_name
Tet2/3-WT_0h_H3K27Ac
strain background
C57BL/6J
age
8-12 weeks
genotype/variation
Tet2/3-WT (Tet2-flox, Tet3-flox, Rosa26-LSL-YFP)
tissue
Spleen
time point
0h
antibody rrid
Abcam Cat# ab4729,RRID:AB_211829
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 1% formaldehyde (ThermoFisher) at room temperature for 10 mins at 1E6 cell/mL in media, quenched with 125 mM glycine, washed twice with ice cold PBS. Cells were pelleted, snap-froze with liquid nitrogen. To isolate nuclei for sonication, cell pellets were thawed on ice and lysed with lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100) for 10 mins at 4°C with rotation, washed once with washing buffer (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and twice with shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.1% SDS). Nuclei were resuspended in 1mL shearing buffer and sonicated with Covaris E220 using 1 mL milliTUBE (Covaris, Woburn, MA) for 18-20 minutes (Duty Cycle 5%, intensity 140 Watts, cycles per burst 200). After sonication, insoluble debris was removed by centrifugation at 20,000 x g. Buffer for chromatin was adjusted with 1 volume of 2x conversion buffer (10 mM Tris-HCl pH 7.5, 280 mM NaCl, 1 mM EDTA, 1mM EGTA, 0.2% sodium deoxycholate, 0.2% Triton-X100, 1% Halt protease inhibitors with 0.1% SDS). Chromatin was pre-cleared with washed protein A dynabeads (ThermoFisher) for 2 hours, incubated with rabbit polyclonal anti-H3K27Ac (ab4729, Abcam) and protein A dynabeads overnight (all procedures were at 4°C with rotation). Bead-bound chromatin was washed twice with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS), once with high salt wash buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and once with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Chromatin was eluted from beads with elution buffer (100 mM NaHCO3, 1% SDS, 1 mg/mL RNaseA; Qiagen) twice for 30 mins each at 37°C with constant shaking. NaCl and proteinase K (Ambion) were added to the eluted chromatin at concentrations of 250 mM and 0.5 mg/mL, respectively, and de-crosslinked at 65°C overnight with constant shaking. DNA was purified with Zymo ChIP DNA Clean & Concentrator-Capped Column (Zymo Research, Irvine, CA). Library was prepared with NEB Ultra II library prep kit (NEB) following manufacture's instruction. ChIP-seq, 50bp single end with HiSeq2500