ChIP-seq Protocol for Chromatin Binding Factors

Cells were fixed in medium or phosphate buffered saline with 1% formaldehyde at room temperature for 10 min. ChIP experiments were performed as described^{46 (link)}. ChIP-seq libraries were prepared using NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (New England BioLabs, E6240). Rabbit polyclonal antibody for Rad21 (1:1000 dilution for western blot; 2.5 ug/million cells for ChIP-seq) was obtained from Eurofins Genomics and has been described in^{47 (link)}. Antibodies for MAU2 (ab46906, 2.5 ug/million cells as dilution) and SA1 (ab4457, 2.5 ug/million cells) were from Abcam. Antibodies for TAF1 (A303-505A, 2.5 ug/million cells) and AFF4 (A302-538A, 2.5 ug/million cells) were from Bethyl Laboratory. CTCF (07-729, 2.5 ug/million cells) antibody was from Merck Millipore. Antibodies (2.5 ug/million cells) for unphosphorylated Pol2 (CMA601), Pol2ser2 (CMA602) and H3K27ac (CMA309) were kindly provided by Dr. H Kimura (TITech), which were used in previous studies^{17 (link),48 (link)}. Antibody for CBP (606402, 2.5 ug/million cells) was from BioLegend. Antibodies for P300 (sc-585, 2.5 ug/million cells) and Med1 (sc-5334, 2.5 ug/million cells) were from Santa Cruz Biotechnology.

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Wang J., Bando M., Shirahige K, & Nakato R. (2022). Large-scale multi-omics analysis suggests specific roles for intragenic cohesin in transcriptional regulation. Nature Communications, 13, 3218.

Publication 2022

NEBNext ChIP-seq Library Prep Master Mix Set for Illumina Antibodies for ab4457

Antibodies Antibody Cells Chip Chip seq Ctcf Dilution Formaldehyde Library Med1 P300 Phosphate Rabbit Saline Western blot

Corresponding Organization :

Other organizations : The University of Tokyo

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Variable analysis

independent variables

Fixed cells in medium or phosphate buffered saline with 1% formaldehyde at room temperature for 10 min

dependent variables

ChIP-seq library preparation
ChIP-seq sequencing
ChIP enrichment and binding for Rad21, MAU2, SA1, TAF1, AFF4, CTCF, unphosphorylated Pol2, Pol2ser2, H3K27ac, CBP, P300, and Med1

control variables

Rabbit polyclonal antibody for Rad21 (1:1000 dilution for western blot; 2.5 ug/million cells for ChIP-seq)
Antibodies for MAU2 (2.5 ug/million cells), SA1 (2.5 ug/million cells), TAF1 (2.5 ug/million cells), AFF4 (2.5 ug/million cells), CTCF (2.5 ug/million cells), unphosphorylated Pol2 (2.5 ug/million cells), Pol2ser2 (2.5 ug/million cells), H3K27ac (2.5 ug/million cells), CBP (2.5 ug/million cells), P300 (2.5 ug/million cells), and Med1 (2.5 ug/million cells)

positive controls

Previous studies using the same antibodies for unphosphorylated Pol2, Pol2ser2, and H3K27ac (references 17 and 48)

negative controls

None specified

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