NextSeq 500 sequencing; ChIP-seq of ZFP30-HA and KAP1 in mouse 3T3-L1 cells during adipogenic differentiation
ChIP-seq of ZFP30-HA and KAP1 in mouse 3T3-L1 cells during adipogenic differentiation
Chromatin immunoprecipitation was performed as previously described (Blecher-Gonen et al., 2013). Briefly, 2*15 cm dishes of cells were cross-linked with 1% formaldehyde at room temperature for 10 minutes. The cross-linking was quenched by fresh glycine at a final concentration of 0.15 M. The cells were then harvested and lysed in a series of ice-cold lysis buffer to collect the nuclei. 3T3-L1 cells were induced to differentiation at day 0 (2 days post-confluence) by adding the induction medium, which is complete culture medium supplemented with the MDI cocktail (1μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, and nM insulin, all from Sigma, Saint-Louis, MO). At day 2, induction medium was removed and maintenance medium (complete culture medium supplemented with 167 nM insulin) was added. 1 mL of nuclei suspension was transferred to Covaris 1 mL glass tube and sonicated at 10% Duty factor, peak incident power 140W, and cycle per purst 200 for 20 min in Covaris E220. 20 μl of the sonicated chromatin was de-cross-linked and the DNA was extracted for quality control. The majority of the DNA is around 200 bp. Those passed the quality control were divided into 25 μg of DNA each aliquot and 1 μl was kept as input. 75 μl of magnetic protein G beads coupled with 10 μg of anti-HA (ab9110, Abcam), anti-KAP1 (KAP1-a1: ab10483 (Abcam), KAP1-a2: ab22553 (Abcam)), anti-p300 (sc-585, Santa Cruz) or anti-IgG control antibody (12-371, Millipore) was incubated with each aliquot of chromatin overnight at 4 C. The ChIPmentation method (Schmidl et al., 2015) was used to generate the ChIP-seq library. In brief, the chromatin precipitated by the beads was subjected to Tn5 transposase-mediated tagmentation and adaptor ligation at one single step. Then the tagmentated chromatin was digested with Proteinase K and RNase, then de-cross993 linked by heating at 65ÅãC overnight. The DNA was then purified using the Agencourt AMPure XP beads, and amplified using the KAPA HiFi Hotstart 994 ReadyMix kit (KAPA Biosystems) and Nextera Index (Illumina) primer sets for 15 cycles.
Experimental Factor: immunoprecipitate
anti-IgG
Experimental Factor: time
2