title: GSM6268526: Stage 9 animal cap ATAC rep 2 size selected; Xenopus laevis; ATAC-seq
library_construction_protocol: ATAC procedure was based on Esmaeili et al Dev Biol 2020. Dejellied embryos were incubated in MR/3 (33 mM NaCl, 0.6 mM KCl, 0.67 mM CaCl2, 0.33 mM MgCl2, 1.67 mM HEPES, pH 7.8) at 23C until desired NF stage and then devitellinized with 1 mg/mL pronase. Ectodermal explants (animal caps) were dissected using watch-maker forceps in 0.7x MR. Two caps were used per transposition reaction. Caps were washed in ice cold PBS and centrifuged at 500xg in 4C for 5mins twice. Caps were washed again and lysed in 50 µl of RSB buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630) with a clipped P200 pipet. The lysate was pelleted at 500xg in 4C for 10 min and resuspended in in 47.5 µl TD buffer (10 mM Tris pH 7.6, 5 mM MgCl2, 10% dimethylformamide) and 2.5 µl of 3 µM Tn5 transposome (Addgene #112112). Nuclei were transposed with gentle shaking for 1 hr at 37C before adding 2.5 µl proteinase K and incubating overnight at 37C. Transposed DNA was purified using EconoSpin Micro columns (Epoch) and amplified using 25 µM indexed Nextera primers with Thermo Phusion Flash master mix for 12 cycles. The amplified library was column cleaned and verified by Qubit dsDNA high sensitivity and Fragment Analyzer and sequenced multiplexed paired end at the Health Sciences Sequencing Core at Children's Hospital of Pittsburgh. For reps 1 and 2, after initial sequencing, libraries were subsequently size selected on an agarose gel to enrich for 150-250 and 250-600 bp fragments and resequenced pooled. For reps 3 and 4, libraries were only size selected for 150-250 bp.