ATAC-seq of bovine embryos
Oocytes or embryos (a minimum of 500 cells) were treated with pronase (10 mg/ml) to completely remove the zona pellucida and washed with SOF-Hepes on a warming plate. Cells were then transferred to 1 ml cold SOF-Hepes, and centrifuged at 500 rcf, 4C, for 5 min. Morulae were vortexed for 3 min in cold ATAC-seq lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Cell pellets were then resuspended in 1 ml cold ATAC-seq cell lysis buffer and centrifuged at 500 rcf, 4C, for 10 min. Nuclear pellets were then resuspended in 50 l transposition reaction mix (25 l TD buffer (Nextera DNA Library Prep Kit, Illumina), 2.5 l TDE1 enzyme (Nextera DNA Library Prep Kit, Illumina), 22.5 l nuclease-free H2O) and incubated for 60 min at 37C, shaking at 300 rpm. The transposase, which is loaded with Illumina sequencing adapters, cuts DNA where it is not sterically hindered and simultaneously ligates adapters, effectively producing a library in one incubation step. Transposed DNA was purified with the MinElute PCR purification kit (Qiagen, Hilden, Germany) and eluted in 10 l buffer EB. Libraries were then PCR amplified: 50 l reactions (25 l SsoFast Evagreen supermix with low ROX (Bio-Rad, Hercules, CA), 0.6 l 25 M custom Nextera PCR primer 1, 0.6 l 25 M custom Nextera PCR primer 2 (with barcode; Supplementary Table 2), 13 l nuclease-free H2O, and 10 l eluted DNA) were cycled as follows: 72C for 5 min, 98C for 30 s, and then thermocycling at 98C for 10 s, 63C for 30 s and 72C for 1 min. Libraries from GV oocytes, 2-cell, and 4-cell embryos were thermocycled for 13 cycles, and 8-cell and morulae libraries were thermocycled for 11 cycles. PCR-amplified libraries were again purified with the MinElute PCR purification kit (Qiagen, Hilden, Germany) and eluted in 10 l buffer EB. Libraries were then evaluated for DNA concentration and nucleosomal laddering patterns using the Bioanalyzer 2100 DNA High Sensitivity chip (Agilent, Palo Alto, CA). Expected nucleosomal laddering was evidenced by the presence of both small fragments, corresponding to hyper-accessible DNA that was frequently transposed, and larger fragments, corresponding to DNA that was wrapped around one or more nucleosomes. This study focused on mapping open chromatin; therefore, the sub-nucleosomal length fraction of each library (150-250 bp) was size selected using the PippinHT system (Sage Science, Beverly, MA) with 3% agarose cassettes. Size-selected libraries were run on a Bioanalyzer DNA High Sensitivity chip to confirm size-selection and determine DNA concentration. Final libraries were then pooled for sequencing on the Illumina NextSeq platform to generate 40 bp paired end reads.