GSM5931561: TELS hybrid S33-HiC_rep1; Xenopus tropicalis x Xenopus laevis; Hi-C
Male Xenopus tropicalis were injected with 150 U HCG. After 2 hours, two X. tropicalis testes collected in 1 ml 1xMBS and homogenized using razor blades. The solution was filtered with 40um strainer and its volume was brought down to 14ml. Then, through twice 180G horizontal centrifugation, we can obtain pure sperm in the supernatant. Ovulation and cross-fertilization were carried out according to the protocol described previously with some modification (Gibeaux., 2018). Briefly, Female Xenopus laevis were injected with 500 U HCG. After 12 hours, male Xenopus tropicalis were injected with 150 U HCG. After 2 hours, X. laevis females were squeezed gently to deposit eggs onto clean 9 cm Petri dishes and two X. tropicalis testes collected in 0.5 ml 1xMBS and homogenized using razor blades. Any liquid in the Petri dishes was removed and the per dish eggs were fertilized with mix solution containing 0.1 ml of sperm and 0.9 ml H2O. Eggs were swirled in the solution to individualize eggs as much as possible and incubated for 10 min. Jelly coats were removed with a 2% cysteine solution (add NaOH adjust the pH to 7.8-8.0). After extensive washing (at least four times) with 0.1xMBS, embryos were incubated at 23 °C. 1-2 millions sperm（or 50-200 X.laevis x X.tropicalis embryos） were cross-linked with 1% formaldehyde for 10 min using vacuum infiltration. (Human K562 cells were added as spike-in control.) Isolated nuclei were digested with 80U of DpnII (NEB, R0543L) at 37°C for 4 hrs. Restriction fragment overhangs were marked with biotin-labeled nucleotides. After labeling, chromatin fragments in proximity were ligated with 4000U T4 DNA ligase for 6 hrs at 16°C. Chromatin was reverse cross-linked, purified, and precipitated using ethanol. Biotinylated ligation DNA was sheared to 250-500bp fragments followed by pull-down with MyOne Streptavidin T1 beads (Life technologies, 65602). Immobilized DNA fragments were end-repaired, A-tailed, and ligated with adaptors. Fragments were then amplified with the Q5 master mix (NEB, M0492L). Hi-C libraries were sequenced on the Illumina HiSeq X10 platform (PE 2×150 bp reads).