EXPERIMENT_TITLE: GSM7921287: Hi-C_Hae3_Alu1, replicate 1; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: To lyse cell pellet, we prepared 2 ml of ice-cold Hi-C lysis buffer (10 mM Tris·Cl, pH 8.0, 10 mM NaCl, 0.2% Igepal) with protease inhibitors. The cross-linked cells (10M) were resuspended with 1 ml of Hi-C lysis buffer. The cell lysis was incubated on ice for 20 min with rotation at 4°C and then centrifuged at 1500g for 3 min at 4 °C to discard supernatant. The pellet was washed once with 0.5 ml ice-cold Hi-C lysis buffer. The pellet was resuspended with 250 μL 0.5% SDS and incubated for 7 min at 62 °C. 725 μL of water and 125 μL of 10% Triton X-100 was added to quench the SDS. The mix was incubated at 37°C for 5 min and then was spun down at 1500g for 3 min at room temperature to discard supernatant. The pellet was washed once with 1ml 1× PBS (0.1% Tritonx-100) and transferred to a new tube. Then, 500 μL 1× CutSmart (0.1% Tritonx-100) with 200U Hae3 (10M cells) was added, and the chromatin was digested at least 4hr at 37 °C with rotation. The mix was span down at 1000g for 3 min at 4 °C to discard supernatant. To fill in restriction fragment overhangs and mark DNA ends with biotin, 400 μL of Klenow (3`-5`exo-) solution (40 μL NEB buffer2, 8 μL 10 mM dATP, 40 μL 10% TritonX-100, 308 μL H2O, and 4 μL Klenow (3`-5`exo-, 5U/μL, NEB M0212L) was added to the digested chromatin and incubated at 37°C for 1hr with rotation. The mix was centrifuged at 1000g for 3 min at 4 °C to discard supernatant. The pellet was washed once with 1ml 1× PBS (0.5% Tritonx-100) and once with 200 μL 1× T4 DNA ligase buffer. The pellet was resuspended with 1200μL of proximity ligation solution (946 μL H2O, 120 μL T4 DNA ligase buffer, 120 μL 10% TritonX-100, 8 μL T4 DNA ligase (400U/μL, NEB M0202L), 6 μL 20 mg/ml BSA, 4 μL Bridge linker (100 ng/μL)). The mix was rotated at room temperature for 4hr or 4 °C overnight.  Here, we saved ¼ of the ligation products for HiC and the remaining ¾ for H3K27ac HiChIP (see below). To remove open-DNA and linkers, ligation products were centrifuged at 1000g for 3 min at 4 °C to discard supernatant. Then, 700 μL Exonuclease mix (70 μL 10×lambda cut buffer, 3 μL lambda exonuclease (5U/μL, NEB M0262S), 3 μL exonuclease I (20U/μL, NEB M0293S), 624 μL H2O, 3.5 μL 10% Tritonx-100) was added and rotated at 37 °C for 1hr. The mix was centrifuged at 1000g for 3 min at 4 °C to discard supernatant. The pellet was washed once with 1ml 1× PBS (0.1% Tritonx-100). To remove protein and purify DNA, the pellet was resuspended in 500 μL 1×Protease lysis buffer (10mM Tris·HCl (pH 8), 100mM NaCl, 25mM EDTA, 0.5%SDS, 0.2mg/ml protease K). The sample was incubated for 5-10h at 65 °C. The mix was centrifuged at 12,000g for 5 min at RT and supernatant was saved into a new tube. Then, 2×volume 100% ethanol was added to the supernatant and placed at -20°C for 10 mins. The mix was centrifuged at 12,000g for 10 min at 4°C to pellet the DNA. The DNA pellet was washed with 70% ethanol and spun down at 12,000g for 10 min at 4°C. The DNA pellet was dissolved in 140 μL 10 mM Tris-HCl (pH 8) buffer and incubated at 65°C for 5 min. Then, the solution was centrifuged at 12,000g for 5 min to remove melanin. To make biotinylated DNA suitable for high-throughput sequencing, DNA was sheared to 300-400 bp with a Covaris M220 (Use 20ug DNA in a 130 μL Covaris microtube. Peak incident power 50; duty factor 20%; cycle per burst 200; time: 65 seconds). To capture biotinylated DNA fragments, 30μL Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602) was washed once with 500 μL 1×TWB (5 mM Tris-HCl pH 7.5, 1M NaCl, 0.5 mM EDTA, 0.05% Tween20), once with 500 μL 1× binding and washing buffer (B&W, 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA), and then the beads were resuspended using 200 μL 2× B&W and added to the 200 μL biotinylated DNA. The mix was incubated for 30min at RT with rotation. Beads were separated magnet and the supernatant was discarded. Beads were washed 2 times with 600 μL 2×SSC/0.5% SDS, once with 600 μL of 1X TWB and transferred to a new tube. The beads were heated on a Thermomixer at 55°C for 2 min with mixing, and then resuspended with 50 μL 5 mM Tris (pH 8) and transferred to a new PCR tube.To enrich H3K27ac mediated interactions, sonicated chromatin was centrifuged at 12,000g for 15 min at 4°C and supernatant was transferred to a new tube. Then, 10μL of protein G beads (10M cells) were added to the chromatin and incubated for 1 hour at 4°C with rotation. Beads were separated on a magnetic stand and supernatant was transferred to a new tube. Then, 7.5 μg of H3K27ac antibody (Abcam, ab4729) was added to the tube and incubated 2 hours at 4°C on a rotator. Then, 50μL of protein G beads (10M cells) was added to the sample and incubated for 2 hours at 4°C on a rotator. Beads was collected using a magnetic stand and washed 3 times with 1 mL of high salt buffer (pH 8.0, 20mM Tris, 500mM NaCl, 1mM EDTA, 1mM EGTA, 1% TX-100, 0.1% sodium deoxycholate, 0.1% SDS); 3 times with 1 mL of LiCl wash buffer (pH 8.0, 20 mM Tris, 1mM EDTA, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS); once with 1 mL of TE salt buffer (pH 8.0, 10 mM Tris, 1 mM EDTA, 50 mM NaCl). Beads were resuspended in 100μL of elution buffer (50 mM Tris, 10 mM EDTA, 1% SDS) and incubated at 65°C for 15 minutes to elute. Then, 1 μL of 33 mg/mL RNase A was added to the elution and incubated at 37°C for 30min. Then, 5 μL of 20 mg/mL proteinase K was added to the mix and incubated at 55°C for 30 minutes. Samples were then incubated at 65°C for 5 hours to reverse cross-links. DNA was then purified using Zymo ChIP columns (Zymo, D5102) according to manufacturer's protocol and eluted in 30 μL water. The amount of eluted DNA was quantified by the Qubit kit. To prepare the ChIP DNA for sequencing, the eluted DNA was transferred to a 55 μL sonication tube and sheared to a size of 250-350 bp using Covaris M220 with the following parameters: peak incident power 75; duty factor 10%; cycle per burst 200; Time: 110 seconds). Then, 20 μL Dynabeads MyOne Streptavidin T1 beads (Life technologies, #65602) was added to 200 μL sonicated ChIP DNA and incubated for 20 mins at room temperature with rotation. Beads were collected on a magnet and washed 3 times with 600 μL 2×SSC/0.5% SDS and transferred to a new tube. Then, beads were washed with 600 μL of 1X TWB and the mixture was transferred to a new tube. Beads were heated on a Thermomixer at 55°C for 2 min. The beads were transferred to a new tube and washed 3 times with 600 μL 1×TE. Beads were washed once with 600 μL 5mM Tris (PH 8.0) and resuspended with 50 μL 5 mM Tris, pH 8.0. DNA library preparation was performed on-beads using NEBNext Ultra™ II DNA Library Prep Kit (NEB, E7545S). Hi-C libraries were amplified by KAPA HiFi HotStart ReadyMix (Roche, 07145446001) for 8 cycles. Libraries were purified with AMPure beads (250-500bp) and sequenced on an Illumina HiSeq2000 with 150 bp paired-end reads.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Skin color is highly variable in Africans, yet little is known about the underlying molecular mechanism. We identified 1,157 candidate variants influencing skin pigmentation in indigenous Africans by genome-wide association studies and scans of natural selection based on differentiation in allele frequencies between lightly pigmented southern African Khoesan populations and other darkly pigmented African populations. We applied massively parallel reporter and chromosome conformation capture assays to identify novel regulatory variants and their target genes related to skin pigmentation in melanocytic cells. We identified 165 SNPs showing strong differential regulatory activities between alleles. Combining CRISPR-mediated genome editing, transcriptome profiling and melanin assays, we identified causal regulatory variants impacting pigmentation near MFSD12/HMG20B, MITF, OCA2, and DDB1/CYB561A3/TMEM138. We identified CYB561A3 as a novel gene regulating pigmentation by impacting genes involved in oxidative phosphorylation and melanogenesis. Our results broaden our understanding of the genetic basis of human skin color diversity and human adaptation. To decipher the target genes of the MFVs, we performed Hi-C and H3K27ac HiChIP assays in MNT1 cells. Hi-C is a high-throughput method for detecting chromatin interactions at whole genome scale and is often used to identify topologically associating domains (TADs) in the nucleus. H3K27ac HiChIP can identify chromatin interactions enriched for H3K27ac, a histone modification associated with active promoters and enhancers. We performed bridge linker mediated Hi-C  and H3K27ac HiChIP using double (Hae3_Alu1) as well as single (Hae3) enzyme digestion in MNT-1 cells. Overall design: Hi-C and H3K27ac HiChIP in MNT-1 cells
SAMPLE_TITLE: Hi-C_Hae3_Alu1, replicate 1
SAMPLE_SAMPLE_ATTRIBUTES_SAMPLE_ATTRIBUTE: [{"TAG": "source_name", "VALUE": "MNT-1"}, {"TAG": "cell line", "VALUE": "MNT-1"}, {"TAG": "cell type", "VALUE": "melanoma cells"}, {"TAG": "genotype", "VALUE": "None"}, {"TAG": "antibody", "VALUE": "none"}, {"TAG": "time", "VALUE": "None"}, {"TAG": "geo_loc_name", "VALUE": "missing"}, {"TAG": "collection_date", "VALUE": "missing"}]
Pool_Member_@attributes_sample_title: Hi-C_Hae3_Alu1, replicate 1
RUN_SET_RUN_Pool_Member_@attributes_sample_title: Hi-C_Hae3_Alu1, replicate 1
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