NextSeq 500 paired end sequencing; ChIP-seq of T/Brachyury in mouse embryonic stem cells in the process of disruption of naive pluripotency caused by T induction
ChIP-seq of T/Brachyury in mouse embryonic stem cells in the process of disruption of naive pluripotency caused by T induction
T_ChIP_p
T/Brachyury-overexpressed ESCs (equivalent to 1 x 10^7 cells) were dissociated with TrypLE Express, washed, and collected by centrifugation. For crosslinking, the pellet was resuspended in PBS containing 1% formaldehyde, incubated for 10 min, and quenched with 125 mM glycine. The fixed cells were resuspended in 1 ml of cold lysis buffer 1 (20 mM Tris, 10 mM NaCl, 2.5 mM MgCl2, 0.2% NP-40, and 1 mM phenylmethylsulfonyl fluoride [PMSF], pH 7.5), rotated for 10 min at 4 degree, and pelleted by centrifugation at 1,500 xg for 5 min. The pellet was resuspended in 1 ml of lysis buffer 2 (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 1 mM PMSF [pH 8.0]) and rotated for 10 min at 4oC. The remained nuclei were pelleted by centrifugation at 1,500xg for 5 min at 4oC and lysed in 400 ml of lysis buffer 3 (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF). The lysed nuclei were sonicated using a Bioruptor with high power output for 10 cycles (30 s ON and 30 s OFF at 0 degree) x4. Protein-DNA complexes were then immunoprecipitated for 15 hr at 4 degree using 2 mg of anti-V5 antibody (V8012, Sigma-Aldrich) bound to 50 ul of Dynabeads (M-280 Sheep anti-Mouse IgG, Invitrogen). Immunoprecipitates were sequentially washed with 200 ml each of low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, and 150 mM NaCl [pH 8.0]), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, and 500 mM NaCl [pH 8.0]), RIPA buffer (50 mM HEPES-KOH, 0.5 M LiCl, 1 mM EDTA, 0.5% Na-Deoxycholate, and 1% NP-40 [pH 7.4]), and TE buffer (10 mM Tris and 10 mM EDTA [pH 8.0]). For collection of the protein-DNA complexes, beads were resuspended with 50 ml of elution buffer (100 mM NaHCO3, 1% SDS, and 10 mM dithiothreitol). The crosslink for both the immunoprecipitated and input DNA fragments was reversed by incubating at 65 degree for 5 hr, followed by the Proteinase K digestion at 50 degree. The collected DNA was purified by using a Nucleospin gel and PCR clean-up (MACHEREY-NAGEL) and dissolved with TE buffer. To prepare the DNA-library for massively parallel sequencing, sonicated DNA fragments were end-repaired, ligated to sequencing adapters, and amplified by using the NEBNext Ultra II DNA library Prep Kit for Illumina (E7645).