Table 2.

Representative sonication conditions for Subheading 3.2

Cell type	Example	Starting quantity	Suggested sonication time (min)
Tissue	Mouse testis	90 mg	30–35
Suspension aggregates	Neurospheres, embryoid bodies	5×10 cm plates	30–35
Adherent colonies	hESC	5×6-well plates	25–30
Adherent monolayers	Tet21N, mouse embryonic fibroblasts	5×15 cm plate	20–25

Starting quantities of cells can be divided into multiple IPs with different antibodies after sonication (generally 30–50 mg tissue per IP or 2–3 × 10 cm plates of cells per IP). Times given are optimized for ChIP-seq, which requires sonication to a size of 200–500 bp. ChIP without sequencing (using end-point PCR or qPCR to examine specific target genes) can tolerate somewhat larger fragments, so use about one-third less time. All sonications are performed in 100–300 μl total chromatin volume in a 1.5 ml tube using a Diagenode Bioruptor set to sonicate in cycles of 30 s high, 1 min off. Sonication is performed in 15 min increments, incubating the chromatin on ice for 5 min between increments