title: GSM8941250: Sperm pronuclei, Interphase, H2AZ, CnT-BS, biol rep 2; Xenopus laevis; OTHER
library_construction_protocol: For sperm pronuclei: Samples were then diluted 1:4 with 1xDB3 buffer [10mM HEPES-KOH pH 8.0, 100mM NaCl, 0.5 mM spermine, 0.5 mM spermidine, 1 mM ASEBF (GoldBio Cat#A-540-1), 1x ROCHE cOmplete™ protease inhibitor cocktail (Cat# 11697498001), 200 mM Sucrose, 0.1% Tween-20], layered onto 1 mL of 60SDB3 [1xDB3 + 60% sucrose] and centrifuged at 4ᵒC, 10,000 rpm for 45 min (Beckman Coulter SX241.5 rotor) to collect nuclei. For XTC-2: Nuclei isolation was performed at 4 ᵒC using the Nuclei EZ Prep kit (Millipore Cat# NUC101-1KT) as per manufacturer's protocol and resuspended in 1x CnT-WB. For both: unmethylated lambda DNA was added to extracted nuclei at a final concentration of ~1% total DNA mass to assess for bisulfite conversion efficiency. Libraries were prepared as specified in (Li et al. 2021; https://doi.org/10.1016/j.crmeth.2021.100118) using Illumina Nextra i5/i7 adapter primers. Libraries were amplified using 14 cycles on the thermocycler. Amplified samples were then purified using the MinElute PCR purification kit (Qiagen Cat# 28004), concentration measured using qPCR, pooled, and size selected at 200-1000bp in length on a 2% Agarose E-Gel EX (SYBR Gold II). Libraries were validated using the Agilent High Sensitivity DNA kit.