truChIP Ultra-Low Chromatin Shearing Kit with Formaldehyde

Protocol tips

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	24 h after silencing, the cells were washed with PBS and complete MEM media was added to each well and left for additional three days, after which ChIP was performed using truChIP™ Ultra Low Cell Chromatin Shearing Kit (Covaris, Inc., Woburn, Massachusetts).

Protocol tips
24 h after silencing, the cells were washed with PBS and complete MEM media was added to each well and left for additional three days, after which ChIP was performed using truChIP™ Ultra Low Cell Chromatin Shearing Kit (Covaris, Inc., Woburn, Massachusetts).

Publication protocol

For chromatin immunoprecipitation, the calvarial cells were plated at a seeding density of 2 × 105 cells and left undisturbed for 24 h prior to silencing using lentivirus directed towards shC or shGATA4 as described above. 24 h after silencing, the cells were washed with PBS and complete MEM media was added to each well and left for additional three days, after which ChIP was performed using truChIP™ Ultra Low Cell Chromatin Shearing Kit (Covaris, Inc., Woburn, Massachusetts). The histone 3 lysine 4 dimethylation (H3K4me2, catalog #07–030) and histone 3 lysine 27 trimethylation (H3k27me3, catalog #17–622) antibodies were purchased from Millipore (Burlington, Massachusetts). The antibodies were incubated with protein A magnetic beads (Dynabeads, ThermoFisher Scientific catalog # 10002D) and then added to chromatin.

The calvarial cells from 2-day-old Flag-bio pups were grown in α-MEM media with 10% fetal bovine serum. Once confluent, the cells were fixed with 16% formaldehyde for 5 min, and the excess formaldehyde was quenched with 10× glycine for an additional 5 min. 100 μL of streptavidin-coupled Dynabeads (ThermoFisher Scientific, catalog # 65–601) were used for each ChIP reaction. The sheared chromatin was pre-cleared for one hour with 100 μL of protein A magnetic beads (Dynabeads, ThermoFisher Scientific catalog # 10002D). The precleared chromatin was then transferred to the streptavidin beads and incubated overnight at 4 °C. After the incubation, the samples were sequentially washed with cold SDS wash buffer, high salt buffer, LiCl buffer, and TE buffer [19]. To reverse the cross-links the IP beads and the input were resuspended in elution buffer and placed on water bath at 70 °C overnight followed but incubation on heat block for 2 h at 55 °C and an additional 1 h at 37 °C with proteinase K and RNAse, after which the DNA was purified using QIAquick PCR purification kit (Qiagen, Valencia, CA, USA) and was validated by qPCR with primers listed in Supplemental Table 1. Each ChIP was performed in biological triplicates. ChIP DNA was sequenced on an Illumina NextSeq 500. The sequences were aligned to the mouse genome (mm10) using BWA [20] and peaks were called using HOMER [21] with a P value of 0.001.

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