GSM6735714: E11.5 limb bud, ZRS-LacZ, enhancer-capture HiC; Mus musculus; Hi-C
E11.5 embryos were collected for dissection using approved institutional protocols. Tissue from multiple embryos was pooled together in the same collection tube, and at least two separate collection tubes were collected for each tissue for biological replication. Tissues were incubated with collagenase (Gibco) in a thermomixer at 37 ℃ until the cells were dissociated, about 10 to 20 min. The single-cell solution was fixed by adding formaldehyde (Sigma-Aldrich) to a final concentration of 2% at RT for 10 min as described previously. Ice-cold glycine solution was added to a final concentration of 200 mM to quench crosslinking. Cells were then resuspended in cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1.15% Triton X-100 and 1X protease inhibitor cocktail (Thermo Scientific)) and incubated on ice for 15 min. Nuclei pellets were obtained by centrifuge at 750 g for 5 min at 4 ℃, followed by snap-freezing and storage at -80 ℃. Frozen nuclei pellets (2-6 million) were thawed on ice, followed by adding SDS and Triton X-100 to remove non-crosslinked proteins and sequester SDS, and digested using DpnII (NBE) overnight at 37 ℃. The ends of restriction fragments were labeled with biotinylated dCTP and ligated at room temperature for 4 hours. After de-crosslinking and precipitation, ligated products were sheared using a Covaris sonicator (duty cycle: 10%, intensity: 5, cycles per burst: 200, treatment time: 180 s in total) to an average fragment size of 200bp. The ligated sheared 3C libraries (10-12 ug for each replicate) were pulled down using Streptavidin Dynabeads (Thermo Scientific) to get rid of unligated fragments, followed by end repair, adaptor ligation and library amplification according to modified Agilent SureSelectXT protocol. The ecHi-C library was created by performing a target-enrichment protocol using capture RNA probes according to Agilent SureSelect XT protocol with an input amount of 750 ng of Hi-C library per sample. Following hybridization to the RNA oligo library, each ecHi-C library was amplified using 13 cycles and post amplification libraries were size selected at 200-500bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were sequenced (paired-end 100 or 150 bp) using Illumina NovaSeq 6000 to enrich enhancer-centric interactions yielding a total of 1 billion unique paired-end reads.