title: GSM3619418: HiC_ED_rep2_a; Drosophila melanogaster; Hi-C
library_construction_protocol: Hi-C experiments from EDs and 16-18h embryos were conducted as previously described in Ogiyama, et al. 2018. For larval Hi-C triplicates, ~200 EDs were quickly dissected (<1h) from wandering larvae at R.T in Schneider's insect medium before being directly processed. Briefly, formaldehyde was added to reach a 2% final concentration and the samples were homogenized for 3 minutes using a tight tenbrock at R.T and transferred on a rotating wheel (total time for fixation is 10min). Then, cell membrane was lysed on ice and nuclei were subjected to DpnII treatment O/N at 37°C. The next day, restriction sites were end-repaired and biotinylated using Klenow (NEB, M0210) and biotin-14-dATP (Life Tech., 19524-016) before being re-ligated in situ using T4 DNA ligase (NEB, M0202). Then, proteins were degraded with proteinase K before O/N de-crosslinking at 68°C and subsequent DNA purification using Phenol/chloroform and ethanol precipitation. Purified DNA was sheared with a LE220 Covaris sonicator (volume: 130µl, fill lvl: 10, duty cycle: 15, PIP: 500, cycles/burst: 200, time: 58s). Finally, sonication efficiency was checked on an agarose gel. DNA fragments were size-selected using AMPure XP beads (300-500bp), and ligation fragments containing biotin were immobilized on MyOne Streptavidin T1 beads (Life Technologies, 65602). Biotin was removed from unligated ends, and pulled-down DNA fragments were end-repaired and a-tailed before NEXTflex (Bioo Scientific, 514101) adaptors ligation. Finally, libraries were PCR amplified (10-12 cycles) using NEBNext® Ultra™ II Q5® Master Mix (New England Biolabs, M0544L) and DNA was then double-size selected using AMPure XP beads (Agencourt, Cat.N: A63881) in order to isolate fragments between 300 and 800bp.
attributes.tag: GEO Accession
attributes.value: GSM3619418