GSM2814028: Zebrafish 4hpf_4C_view4_AC; Danio rerio; OTHER
Carefully staged embryo's were dechorionated, deyolked and made single cell in three consecutive steps. First embryos were dounched and spun down at 500 rcf at 4 degrees. The precipitant was thereafter incubated in 2 ml of TRIPLE (life, Cat# 12605-010) for 5 min at RT after which 10% end concentration FBS was added. This solution was filtered using a 100uM cell strainer (Sigma, Cat# CLS431752-50EA and cells was spun down at 500 rcf at 4 degrees to collect the single cells. In brief after obtaining a single cell suspension, crosslinking, primary digestion with DPNII and ligation the DNA was reverse crosslinked o/n at 65C. DNA was isolated by phenol/chloroform extraction and subsequently digested with a 2nd restriction enzyme (CSP6I). Digested DNA was again ligated, but this time under diluted conditions (10ml). DNA was precipitated with 1/10 volume 3M sodium acetate and 1 volume isopropanol. DNA was quantified using QUBIT (thermo fisher). 4C pcr was done using NEB-Next High-Fidelity (NEB,M0541) in 4 separate PCR reactions using ~1-200ng per PCR
GEO Accession
GSM2814028