rex-32(y572) rex-33(y743) rex-14(y738) rex-47(y671) rex-8(y737) rex-43(y741) rex-48(y742) rex-35(y740) X
Sequenced DNA Library
Embryos were ground with 50 strokes in a 2 mL dounce homogenizer. Sarkosyl was added to a final concentration of 0.1% and chromatin was sheared in an S2 Covaris with duty cycle 20%, intensity 8, and 200 cycles/burst for 30 cycles of 60 sec with 45 sec of rest for a total time of 52 min. The extract was centrifuged at maximum speed for 15 min at 4° C. Supernatant containing approximately 2 mg total protein was incubated with 6.6 μg rabbit polyclonal anti-DPY-27 (rb699) (Chuang et al., 1994), rat polyclonal anti-SDC-3 (PEM4A) (Crane et al., 2015), or random IgG antibodies overnight at 4° C in a volume of at least 500 μl. 50 μl of Protein A Dynabeads (ThermoFisher Scientific, 10002D) were washed in FA buffer three times, added to the immunoprecipitation, and mixed at 4° C for at least 2 hr. Beads were then washed and DNA eluted as in (Kruesi et al., 2013). We performed end repair, A-tailing, ligation of NEXTflex DNA Barcodes, and amplification as in Kruesi et al 2013.