Indexed primers

For ULI-NChIP-seq, 5 ng or 85% of the raw ChIP material was used for library construction using the NEBNext DNA Library Prep Kit and NEBNext Multiplex Oligos for Illumina. In brief, samples were end-repaired, 5′ phosphorylated and dA-tailed, then ligated with NEBNext adaptor. Ligated fragments were amplified using indexed primers (Illumina) and size selected with Ampure XP DNA purification beads.

Early nc14 embryos were placed in ATAC-seq lysis buffer (Buenrostro et al., 2013 (link)) without detergent, with 5% glycerol added. Embryos were then taken out of the freezing solution and placed onto a glass slide which was then put on dry ice for 2 min. Once embryos were completely frozen, the glass slide was removed and embryos were sliced with a razor blade chilled in dry ice. Once sliced embryo halves were moved to tubes containing ATAC-seq lysis buffer with 0.15 mM spermine added to help stabilize chromatin. Embryo halves were then homogenized using single use plastic pestles. IGEPal CA-630 was added to a final concentration of 0.1%. After a 10 min incubation nuclei were spun down and resuspended in water. Twenty halves were added to the transposition reaction containing 25 µl of 2x TD buffer (Illumina), and 2.5 ul of Tn5 enzyme (Illumina) and the reaction was incubated at 37°C for 30 min as in Buenrostro et al. (2013) (link). Transposed DNA was purified using Qiagen Minelute kit. Libraries were then amplified using phusion 2x master mix (NEB) and indexed primers from Illumina. Libraries were then purified with Ampure Beads and sequenced on the Hiseq4000 using 100 bp paired end reads.