GSM5583706: HiC of GFP overexpressing cells rep2; Mus musculus; Hi-C
The somatosensory cortex of in utero electroporated E14.5 mouse embryos was isolated and the electroporated area dissected. Single cell suspension was prepared using the papain-based neural dissociation kit (Miltenyi Biotec, Cat. N: 130-092-628) according to the manufacturer protocol with reduced dissociation times (5 min. instead of 15 min. at 37˚C). Cell number as well as the viability was assessed using the Countess™ II Automated Cell Counter (Invitrogen). Dissociated cells were fixed for 10 min. in 1% Formaldehyde (ThermoFisher, Cat. N: 28908), quenched by addition of Glycine (Invitrogen, Cat. N: 15527013) to a final concentration of 0.2M, washed in PBS with 0.1% BSA, passed through a 40µM cell strainer (ThermoFisher, Cat. N: 15342931) and immediately FAC-sorted at a BD FACSAria Fusion (BD Bioscience) with four lasers (405, 488, 561, 640) using a 100µm nozzle. Sorted cells were lysed with 0.2% Igepal-CA630 (Sigma-Aldrich, Cat. N: I3021), permeabilized with 0.5% SDS (Invitrogen, Cat. N:AM9823) and digested with 200U DpnII (New England Biolabs, Cat. N: R0543) overnight at 37˚C. Subsequently, sticky ends were filled by incubating the nuclei for 4h at RT with DNA Polymerase I (New England Biolabs, Cat. N: M0210) and a biotin-14-dATP (Life Technologies, Cat. N: 195245016) containing nucleotide mix in DpnII buffer followed by proximity ligation for at least 6h at 16°C. Thereafter nuclei were lysed, proximity ligated DNA was reverse-crosslinked overnight at 68˚C followed by a purification by ethanol precipitation and shearing to ~550 bp DNA fragments using a Covaris S220 sonicator. Biotin pulldown was performed by incubation the sheared DNA with MyOne Streptavidin T1 beads (ThermoFisher, Cat. N: 65602) for 30min. at RT followed by on-bead biotin removal and end-repair by incubating the samples for 30 min. at RT in a reacting mix consisting of T4 DNA Polymerase, T4 Polynucleotide Kinase and DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs, Cat. N: M0203, M0201, M0210). Thereafter A-tailing using Klenow Fragment exo-minus (New England Biolabs, Cat.N.: M0212) was performed for 30 min. at 37˚C followed by ligation of NextFlex DNA barcodes (Perkin Elmer, Cat. N.: NOVA-514101) using the NEB Quick Ligase (New England Biolabs, Cat. N: M2200). Between each of the incubation steps the samples bound to the streptavidin beads were washed twice with washing buffer containing 0.05% Tween-20 (Sigma-Aldrich, Cat. N: P9416) and once with the respective buffer of the following reaction. Libraries were amplified on the streptavidin beads using the NEBNext Ultra Q5 II Master mix (New England Biolabs, Cat. N.: M0544) using following program: 98˚C 30s; {98˚C 10s, 65˚C 75s} x10; 65˚C 5min; Hold at 10˚C. After the amplification the streptavidin beads were pelleted and the supernatant was purified twice using 0.7x AMPure XP beads (Agencourt, Cat. N: A63881) to reach an average fragment size of approximately 500bp.