Spleen-HiC
HiC library was generated as described in Nagano et al. (2015) and Wutz et al. (2017), with modifications as described below. 0.2 g of snap-frozen lungfish spleen was powdered and fixed with 1% formaldehyde, 10% FCS/AMEM at room temperature for 10 minutes. The fixation was stopped by adding icecold glycine (125 mM f. c.). Fixed cells were strained with 100 m cell strainer to generate single cells, collected by centrifugation (200 x g for 10 min at 4), and washed with ice-cold PBS twice (200 x g for 10 min at 4). 1  106 cells were collected and incubated in icecold lysis buffer (10 mM TrisHCl pH 8, 10 mM NaCl, 0.2% Igepal CA630, 1% Triton-X100, protease inhibitor cocktail EDTA free) with occasional agitation for 30 min on ice. After centrifugation to pellet the cell nuclei (250 x g for 10 min at 4C), nuclei were washed once with 1.25x NEBuffer 3 (NEB). The nuclei were resuspended in 1.25x NEBuffer3, SDS was added (0.6% f. c.), and the mixture was incubated with agitation (950 rpm for 2 h at 37). Triton X100 was added for quenching the SDS (3.3 % f. c.), and the nuclei were incubated with agitation (950 rpm for 2 h at 37). Restriction digest with DnpII (in 1x DnpII buffer from NEB; 2,000 U per 0.25 million cells) was performed overnight with agitation (950 rpm at 37). Using biotin14dATP, dCTP, dGTP, and dTTP, the DnpII restriction sites were filled in with Klenow (50 U per 0.5 million cells) for 1 h at 37C with repeated agitation (700rpm 10 sec and rest 30 sec for 1 h in a thermal cycler). The ligation was performed overnight at 18 (2000 U of T4 DNA ligase). After ligation, crosslinking was reversed by incubation with proteinase K in SDS buffer overnight at 65C. An additional proteinase K incubation at 65C for 2 h was followed by RNase A treatment and two sequential phenol/chloroform extractions. After DNA precipitation, the DNA was spun down (centrifugation with max-speed for 30 min at 4C). The pellets were resuspended in 20 l TE and the DNA concentration was determined using Qubit 2 device. 20 g of biotinylated DNA was used for the library preparation. The biotin from nonligated fragment ends was removed with T4 DNA polymerase (NEB) for 30 min at 37C and EDTA was added to stop the reaction (10 mM f. c.). DNA was sonicated using the Covaris system to generate DNA fragments with a size peak around 400 bp (Covaris S2 settings: duty factor: 10%; peak incident power: 5 W; cycles per burst: 200; time: 60 sec). After end repair (T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow in the presence of dNTPs in T4 DNA ligation buffer; for 30 min at room temperature), the DNA was purified (Qiagen mini purification kit). A doublesize selection using DNA purification beads (in-home) was performed: First, the ratio of the beads solution volume to DNA sample volume was adjusted to 0.6:1. After incubation for 15 min at room temperature, the sample was transferred to a magnetic separator, the supernatant was transferred to a new Eppendorf tube, while the beads were discarded. The ratio of the bead solution volume to DNA sample volume was then adjusted to 0.9:1 final. After incubation for 15 min at room temperature, the sample was transferred to a magnet. Following two washes with 80% ethanol, the DNA was eluted in Elution buffer (QIAGEN). Biotinylated ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies) on a magnet stand in binding buffer (5 mM Tris pH8, 0.5 mM EDTA, 1 M NaCl) for 30 min at room temperature. dA-tailing was carried on beads: dATP was added with Klenow exo (for 1h at 37C), then the enzyme was heatinactivated (20 min at 65C). After two washes in binding buffer and one wash in T4 DNA ligation buffer, Illumina adapters (Index #10, P-GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG) were ligated onto HiC ligation products bound to streptavidin beads in T4 DNA ligase with slowly rotating (for 2 h at room temperature). After washing twice with wash buffer (5 mM Tris pH 8.0, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween20) and then once with binding buffer, the DNAbound beads were resuspended in a final volume of 20 l 1x NEBuffer2. Captured biotinylated HiC DNA was amplified with 8 PCR amplification cycles (60ul of total PCR reaction with 6ul of biotin-pull down beads with Q5 Ultra PCR mix (NEB)). After PCR amplification, the HiC libraries were purified with DNA purification beads. The concentration of the HiC library was determined by fragment analyzer and qPCR, and the HiC libraries were pairedend sequenced