GSM3271346: Chi-C_siRE_rep2; Homo sapiens; Hi-C
5× 10^7 Kasumi-1 cells were fixed via suspension in 37 ml of RPMI-1640 supplemented with 15% FBS and 2% formaldehyde. The cells were fixed for 10 minutes at room temperature while mixing. 6 ml of 1M glycine (0.125 M final concentration) was added to quench the reaction. Cells were incubated at room temperature for 5 min, followed by 15 minutes on ice. Cells were then pelleted by centrifugation at 400 x g for 10 minutes at 4 °C. The supernatant was discarded and cells were washed in ice cold PBS. The cells were then pelleted and after removing the supernatant, each sample was flash frozen in liquid nitrogen, and stored at −80 °C. Cells were lysed in a tight dounce homogeniser (ten cycles) with 3ml of cold lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal CA-630, one tablet protease inhibitor cocktail (Roche complete, EDTA-free, 11873580001)). Cells were left on ice for five minutes then homogenised another ten times. The lysed cells, in 3 ml lysis buffer, were added to 47ml of lysis buffer and incubated on ice for 30 minutes with occasional mixing.  The chromatin was pelleted at 1800 rpm for 5 minutes at 4°C and the supernatant discarded. The pellet was resuspended in 1ml of 1.25x NEBuffer 2 and split into four. Each sample was then pelleted at 1000 rpm and resuspended in 358 µl of 1.25x NEBuffer 2. 11 µl 10% SDS was added and each tube was incubated at 37°C for 60 minutes, rotating at 950 rpm. Samples were mixed by pipetting up and down every 15 minutes. The SDS was quenched with 75µl 10% Triton X-100 and incubated at 37˚C for 60 minutes, rotating at 950 rpm. During the incubation, samples were mixed by pipetting up and down every 15 minutes. The chromatin was digested overnight with 1500 units of HindIII (NEB R0104T), rotating (950 rpm) at 37 °C. The HindIII cleavage of its restriction site (5' AAGCTT 3') generates a 5' overhang (5'-AGCT-3'), which was repaired to include a biotinylated nucleotide, allowing the enrichment of Hi-C ligation products. To do this, 6µl 10x NEB2, 2µl H2O, 1.5µl 10mM dCTP, 1.5µl 10mM dGTP, 1.5µl 10mM dTTP, 37.5µl 0.4mM biotin-14-dATP (Life Technologies 19524-016), and 10μl 5U/μl Klenow (DNA polymerase I large fragment, NEB M0210L) were added to each tube. Samples were mixed carefully and incubated for 60 minutes at 37˚C, with mixing by pipetting every ten minutes. The digested chromatin mixture was added to a falcon tube with 8 ml of ligation buffer (750µl 10x ligation buffer (NEB B0202S), 75 μl 10mg/ml BSA (NEB B9001S) and 50 μl  1U/μl T4 DNA ligase (Invitrogen 15224-025). These dilute conditions favour ligation of fragments crosslinked within the same chromatin complex (intra-molecular ligation). The tubes were mixed by inversion and incubated overnight at 16 °C. The following day, samples were incubated for a further 30 minutes at room temperature. Crosslinks were reversed and proteins degraded by incubating the tubes over night at 65 ˚C, with 60µl 10mg/ml proteinase K (Roche 03115879001). After the overnight incubation, another 60 µl of 10 mg/ml proteinase K per tube was added, followed by a 2 hour incubation at 65˚C. The reaction mixtures were cooled to room temperature and 12.5 µl of 10 mg/ml RNase A (Roche 10109142001) was added. Samples were incubated at 37˚C for 60 minutes. The DNA in these tubes was purified with two phenol extractions. After ethanol precipitation, the DNA pellets were resuspended in 25µl 1x TE and the four samples were pooled. The Quant-iT assay was used to determine the DNA concentration of the resultant HiC library using a Qubit® 3.0 Fluorometer (Thermo Scientific). To check the library was sufficiently ligated, a 1/10 dilution was resolved on a 0.8% agarose gel. The majority of the library should run at around 10 kb. To confirm that blunt-end ligation was successful, the fact that fill-in and ligation of a HindIII site (AAGCTT) creates a NheI restriction site (GCTAGC) was exploited. A particular ligation product formed by the MYC promoter and a known 1.8 MB long range interaction was amplified with 3C PCR (see table 12 for primer sequences). The same was done for a ligation product formed by the MYC promoter and an adjacent restriction fragment. 200ng of the purified PCR products were split into four and digested with HindIII, NheI or both enzymes, with undigested DNA is used as a control. The resultant DNA samples were run on a 1.5% gel.  To avoid pulling down any non-ligated fragments, biotin was removed from free ends using the exonuclease activity of T4 DNA polymerase. 8 reactions of  5μg of Hi-C library, 0.5μl 10 mg/ml BSA, 5μl 10x NEBuffer 2, 2μl 2.5mM dATP, and 5μl T4 DNA polymerase (NEB M0203L) in a total volume of 50μl were made and incubated at 20˚C for 4 hours. The reaction was stopped by adding 2ml of 0.5M EDTA pH 8.0. Two reactions were pooled to generate four tubes of approximately 10ug DNA. The DNA was purified via phenol:chloroform extraction and ethanol precipitation. Each sample was reconstituted in 130 μl H2O. An E220 focused-ultrasonicator (Covaris) was used to fragment the DNA with the intensity set to 4, the duty cycle set to 10% and with 200 cycles per burst for 55 seconds. After sonication,  the entire volume of each sample was transferred into a fresh Eppendorf tube and 18μl 10x ligation buffer (NEB B0202S),18μl 2.5mM dNTP mix, 6.5μl T4 DNA polymerase (NEB M0203L),6.5μl T4 DNA Polynucleotide kinase (NEB M0201L) and 1.3μl Klenow (NEB M0210L) were added. The reaction was left for 30 minutes at room temperature. Each sample was then split into two and purified with a MinElute column (Qiagen 28004), according to the manufacturer's instructions. Each column was eluted twice with 15 μl TLE (10mM Tris pH8.0, 0.1mM EDTA). To the sheared and end repaired DNA , 5 μl of NEBuffer 2 10x, 11.5 μl dATP 1mM and 3.5 μl Klenow exo- (NEB M0212L) were added. The reaction was incubated for 30 minutes at 37 ˚C. To inactivate the enzyme, each reaction was incubated at 65˚C for 20 minutes, and put on ice immediately afterwards. Fragments between 200 and 650 base pairs were size selected by double-sided SPRI bead (ampure) size selection (0.6x followed by 0.9x), following the manufacturers protocol.  All Hi-C library samples were then pooled and quantified with the Quant-iT™ High-Sensitivity DNA Assay Kit (Invitrogen) and assayed on the Qubit™ Fluorometer (Invitrogen). The biotin marked fragments were immobilized via MyOne Strapavidin C1 DynaBeads (Invitrogen) following manufacturers protocol. The fragments were ligated to illumina paired-end adaptors. The immobilized Hi-C fragments were amplified via PCR, using PE PCR primers 1 and 2 with 7 cycles. The Hi-C library was then purified twice with solid phase reversible immobilization (SPRI) beads (Beckman Coulter Ampure XP beads A63881) with a 1:1.8 DNA to SPRI bead ratio.
GEO Accession
GSM3271346