GSM3939128: LIG3_siLIG1_rep2; Homo sapiens; OTHER
Nuclei were prepared from cells, harvested by trypsinization and washed in PBS + 5 mM EDTA, by incubation in nuclear isolation buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 50 mM EDTA pH 8.0, 0.34 M sucrose, 10% (w/v) glycerol, 0.1% (w/v) Triton X-100) on ice for 10 min. These nuclei were washed once in nuclear isolation buffer and then treated with RNase A at a final concentration of 100 µg/µl in nuclear isolation buffer at 37° C for 15 min. RNase-treated nuclei were resuspended in PBS + 25 mM EDTA prewarmed to 50° C to yield about 700,000 nuclei per 22.5 µl, mixed with an equal volume of molten 1.2% low-melting point agarose in PBS + 25 mM EDTA, and immediately pipetted into a plug mould. After the agarose had solidified, the plugs were incubated twice in Proteinase K solution (1 mg/ml Proteinase K, 1% (w/v) sarkosyl, 125 mM EDTA pH 9.0) overnight at 42° C. The plugs were then washed three times in plug wash buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA) + 1 mM PMSF for 10 min, once in plug wash buffer + 1 mM PMSF for 1 h and once in plug wash buffer for 1 h. Next, individual plugs were transferred to 1.5 ml tubes, rinsed twice with 1 ml 10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA, washed once with 1 ml 10 mM Tris-HCl pH 8.0 + 0.1 mM EDTA for 30 min, twice with 1 ml 0.1 mM EDTA for 15 min, and finally rinsed once with water. After removing as much residual water from the plugs as possible, the DNA was denatured by heating to 95° C for 4 min followed by quenching on slushy ice for 5 min. The plugs were then melted and equilibrated to 37° C, before being supplemented with a ligation mix (6.5 µl 10× T4 ligase buffer, 3.5 µl proximal adaptor (40 µM in 60 mM NaCl), 7 µl water and 3 µl high concentration T4 DNA ligase, NEB) and incubated overnight at 16° C. These reactions were inactivated by addition of EDTA pH 8.0 (10 mM final, in a total volume of 97 µl) and heating to 65° C for 10 min, prior to digestion of the agarose with 3 µl β-agarase at 42° C for 3-4 h. Next, the DNA was fragmented by sonication in a Bioruptor Pico (16 cycles, 30 s on - 30 s off), cleaned up with AMPure beads and eluted in 100 water. This eluate was transferred to a microfuge tube containing 20 µl Streptavidin MyOne C1 Dynabeads (ThermoFisher Scientific, prewashed once with 1 ml 10 mM Tris-HCl pH 8.5, 2 M NaCl) and incubated for 15 min at room temperature with rotation. The beads were washed on a rotating wheel at room temperature: twice with 50 µl 1× SSC buffer each for 5 min at room temperature, once with 25 µl 20 mM NaOH for 10 min and briefly with 100 µl water. The DNA was eluted in 16 µl of water by incubation at 95° C for 5 to 10 min. For second-strand synthesis, the eluted DNA (14.85 µl) was combined with 15 µl Phusion FLASH mix (ThermoFisher Scientific) and 0.15 µl 100 µM oligonucleotide 3790, and incubated at 95° C for 2 min, 60° C for 3 min and 72° C for 2 min. The resulting double-stranded DNA was purified with AMPure beads and eluted in 19 µl water. This elution (17 µl) was A-tailed with 2.3 µl End Prep Reaction Buffer and 1 µl End Prep Enzyme Mix (NEBNext® Ultra™ II DNA Library Prep kit, NEB) for 20° C for 30 min, followed by inactivation at 65° C for 30 min. Distal adaptor (2.25 µl, 40 µM in 60 mM NaCl) was ligated to the A-tailed DNA in the presence of 13.5 µl Ligation Master Mix, 0.45 µl Ligation Enhancer (NEBNext® Ultra™ II DNA Library Prep kit, NEB) and 8.8 µl water at 20° C for 20 min. The ligated DNA was cleaned up twice with AMPure beads, before being eluted in 20 µl water. This DNA (7.25 µl) was mixed with 5 µl 5× Q5 reaction buffer, 2.5 µl 1 µM Illumina P5 primer, 2.5 µl 1 µM Illumina P7 primer, 0.5 µl 10 mM dNTPs, 0.25 µl Q5 DNA polymerase (NEB) and 7 µl water and amplified by PCR, using the following programme: 95° C for 2 min, 8× (95° C for 15 s, 60° C for 30 s, 72° C for 20 s). The amplified DNA was cleaned up twice with AMPure beads, before being eluted in 25 µl water and sequenced following the guidelines supplied by Illumina.  Oligonucleotides:  proximal adaptor: 3898/3899;   distal adaptor: 3791/3792;   3898: CTACACGACGCTCTTCCGATCTNNNNN*N-NH2 (*: phosphorothioate bond, IDT code *; NH2: 3'-amino modification, IDT code /3AmMO/);   3899: PO4-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGTTTT-Bio (PO4: 5'-phosphorylation, IDT code /5Phos/; T-Bio: 3'-biotin-dT, IDT code /3BiodT/);   3790: CGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT;   3791: GACTGGAGTTCAGACGTGTGCTCTTCCGATCT;   3792: GATCGGAAGAGCACACGTCTGAACTCCAGTC;   P5: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT;   P7: CAAGCAGAAGACGGCATACGAGAT(X)6GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT.