GSM8128702: embryo, 50h, rep5; Ascaris suum; OTHER
Ascaris females were collected, and the fertilized embryos (0hr, 1-cell before prenuclear fusion) were harvested and processed as previously described (Wang et al. 2011, Wang et al. 2014). Ascaris 0hr samples were incubated at 30°C with constant shaking for the desired time (from 50hr to 98hr). For all molecular experiments, the eggshells were first de-coated with bleach treatment (0.4 M KOH, 2% sodium hypochlorite) for 1.5 hours at 30°C. Parascaris samples were collected as described (Simmons et al. 2023, Wang et al. 2017). Parascaris eggs were prepared similarly to Ascaris, except the incubation was carried out at 37°C and the embryonation time was shorter (10-14hr). An adapted END-seq protocol was used (Canela et al. 2016, Wong et al. 2021). Embryos were embedded in agarose plugs. Some plugs were treated with the restriction enzymes as internal controls. Plugs were treated with exonuclease VII and exonuclease T. Blunt ends were A-tailed and capped with END-seq adaptor 1 (Canela et al. 2016). Plugs were melted and DNA was sheared to 200-300 bp with a Covaris M220 focused ultrasonicator. DNA fragments containing END-seq adaptor 1 were isolated with Dynabeads MyOne Streptavidin C1. END-seq adaptor 2 was ligated to the sheared ends of the A-tailed DNA fragments. The hairpins within the adaptors were digested with USER, and the DNA was amplified with Illumina TruSeq primers and barcodes. The following modifications were made to the END-seq protocol to capture break ends with different features. For the direct capture method, we excluded the exonuclease VII and exonuclease T treatments. For the all-END protocol, the plugs were treated with T4 polymerase (with dNTPs).