GSM2814020: 24hpf Hi-C rep2 (HiSeq 2500); Danio rerio; Hi-C
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. These cells were subsequently processed following the standard 4C protocol using DPNII as the restriction enzyme (and CSP6I as the second restion enzyme in the case of 4C-Seq). Successful digestion and ligation was confirmed using the agilent TAPEStation.We omitted the usual biotin incorporation and enrichment step due to the low amounts of DNA obtained from the early developmental stages. Instead we generated paired-end deep-sequencing libraries suitable for illumina sequencing after shearing the 3C template to 500-900bp and computationally filtered for ligation events.
GEO Accession
GSM2814020