title: GSM2335037: larvae_rep2; Danio rerio; OTHER
library_construction_protocol: For the 3C library preparation, blood was extracted from 18 dpf zebrafishes or adult fishes. Fishes were euthanized by an overdose of tricaine (ethyl 3-aminobenzoate methanesulfonate, 100 mg/L). For each 3C experiment, a minimum of 107 cells was used. The blood was collected into red cell isolation buffer (0.14 M NaCl, 20 mM HEPES pH 8.0, 5% fetal bovine serum, and 100 U heparin/mL) and fixed with 2% formaldehyde (Sigma) for 10 min at room temperature. The reaction was stopped by adding glycine to a final concentration of 125 mM. All downstream manipulations were performed essentially as described previously (Gavrilov et al. 2008). 5C libraries were prepared in two biological replicates as described previously {Dostie, 2007 #2674}. 5C primer stocks were mixed and diluted to a final concentration of 1.7 nM of each primer. Ten microliters of annealing mixture included the following components: 1 μl of 10×NEBuffer 4 (NEB); 1.7 μl of 5C primer mix; 350 ng of 3C library; 1150 ng of salmon testis DNA (Sigma); and mQ water. Samples were denatured for 5 min at 95°C and annealed for 16 h at 46°C and then ligation in 1×Taq DNA ligase buffer with 10 U of Taq DNA ligase (NEB) was performed for 1 h at 46°C. A total of 2 μl of each 5C library was then PCR-amplified individually with primers to the T7- and T3-tails using KAPA High Fidelity DNA Polymerase (KAPA); 28 PCR cycles were necessary to obtain a sufficient amount of DNA. The PCR mixtures were combined, and the products were purified with a QIAGEN PCR Purification Kit. The DNA was eluted with 50 μl of 10 mM Tris-HCl (pH 8.0) and separated on a 1.8% agarose gel supplied with ethidium bromide; 100 bp fragments were excised from the gel, purified using a QIAGEN Gel Extraction Kit and sequenced on Illumina HiSeq 2000 using single-end 101 bp reads.
attributes.tag: GEO Accession
attributes.value: GSM2335037