ChIP assay on Xenopus tropicalis tadpole intestines was done as described previously (40 (link)) with an antibody against TR (anti-TR) (38 (link)), RNA Polymerase II (abcam, Cambridge, MA), or methylated histone H3K79 (anti-H3K79m2, abcam, Cambridge, MA), and with IgG as a negative control, by using Chromatin Immunoprecipitation (ChIP) Assay kit (Millipore, Burlington, MA). Each sample had three replicas and each replica included at least 5 tadpoles. The immunoprecipitated DNA was analyzed by qPCR with SYBR Green PCR Master Mix on a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, CA). For the analysis of MBD3 TRE, primers 5’-ATGCCCGCCTACTCTTTATTCCTCCAGCTGC-3’ (forward) and 5’-GAGAGAGAGTCAGTGTGGTGGTGGGTCAGA-3’ (reverse) were used. All ChIP experiments were done twice with similar results.
Protocol detail
ChIP Assay on Xenopus Tadpole Intestine
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RNA Polymerase II
Chromatin Immunoprecipitation (ChIP) Assay kit
SYBR Green PCR Master Mix
StepOne Plus Real-Time PCR System
Antibody
Assay
Chromatin immunoprecipitation
Histone
Intestines
Mbd3
Primers
Rna polymerase ii
Sybr green
Tadpoles
Xenopus
Corresponding Organization :
Other organizations : Eunice Kennedy Shriver National Institute of Child Health and Human Development
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Variable analysis
independent variables
- Antibody against TR (anti-TR)
- RNA Polymerase II
- Methylated histone H3K79 (anti-H3K79m2)
- Immunoprecipitated DNA
- IgG as a negative control
- Positive control: Not specified
- Negative control: IgG
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