GSM2399022: Hi-C_NPC_ActbKO2_rep1; Mus musculus; Hi-C
Cells were fixed with 1% formaldehyde for 10 min at room temperature. Glycine was added to 125 mM final concentration and incubated for 5 min at room temperature. Tubes were placed on ice for 15 min. Cells were washed in ice-cold PBS, and 1–2 × 106 fixed cell pellets were used for the following steps. Cell pellets were resuspended in 3C lysis buffer (10 mM Tris-HCl, 10 mM NaCl and 0.2% NP-40, pH 8.0) with 1× protease inhibitor cocktail (Roche) and incubated on ice for 20 min. After centrifugation, pellets were resuspended in 1.2× NEBuffer 2 (New England Biolabs) with 0.3% SDS and incubated for 1 h at 37 °C with constant shaking. Triton X-100 was subsequently added to a 2% final concentration, and samples were incubated for 1 h at 37 °C.  Samples were further incubated overnight at 37 °C with shaking after the addition of HindIII. After centrifugation, pellets were sequentially incubated in 1× NEBuffer 2 with 300 U HindIII for over 8 h at 37 °C with constant shaking in “Fill-in Mix” (1 × NEBuffer 2, 15 µM each of dATP, dGTP, dTTP and 15 µM Biotin-14-dCTP (Thermo Fisher Scientific) with 25 U Klenow (New England Biolabs)) for 45 min at 37 °C and in “Ligation Mix”  (1× T4 DNA ligase buffer (New England Biolabs) with 60 U/l of T4 DNA ligase (Thermo Fisher Scientific)) overnight at 16 °C. After Tris-HCl (pH 7.5) was added at 10 mM, samples were treated with 100 µg/ml RNase A for 30 min at 37 °C, subjected to reverse-crosslinking and proteinase K treatment overnight at 65 °C in the presence of 400 µg/ml proteinase K followed by incubation in 800 µg/ml proteinase K for an additional 2 h.  DNA was extracted by ethanol precipitation after treatment with phenol/chloroform twice and with chloroform once, and resolved in 10 mM Tris-HCl (pH 7.5) (Hi-C DNA). Hi-C DNAs were checked for quantity and quality by Qubit dsDNA HS Assay Kits (Thermo Fisher Scientific) and electrophoresis, respectively. To generate next generation sequencing libraries, Hi-C DNAs were mixed with “T4 DNA pol mix” (1× NEBuffer 2, 1× BSA, 100 µM dATP, 100 µM dGTP and 2.5 U of T4 DNA polymerase (New England Biolabs)) and incubated for 2 h at 12 °C. These DNAs were purified by ethanol precipitation after treatment with phenol/chloroform twice and with chloroform once, and resolved in nuclease-free water. DNAs were next sheared (300–500 bp) using the DNA Shearing System S220 (Covaris) and subjected to size selection using AMPure XP beads (Beckman Coulter). The size-selected DNAs were subjected to biotin pull-down using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific). The sequencing libraries were prepared using Nextera Mate Pair Sample Prep Kit (Illumina) and KAPA Real-Time Library Amplification Kit (KAPA Biosystems), and purification was done with AMPure XP beads. 10 ng of Hi-C DNA was used for construction of a library. To remove biotin from unligated ends, Hi-C DNA was mixed with 50 µl of “T4 DNA pol mix” (1× NEBuffer 2, 1x BSA, 100 µM dATP, 100 µM dGTP, 2.5 U of T4 DNA polymerase (NEB, M0203S)) and incubated for 2 h at 12°C. The sample volumes were brought up to 100 µl by adding 10 mM Tris-HCl (pH 7.5). Samples were extracted once each with phenol/chloroform and chloroform. After EtOH precipitation, the DNA pellets were resuspended in 130 µl of nuclease-free water. DNA samples were sheared (300–500 bp) using the following settings in Covaris S220 (Duty Factor: 5.0, Peak incident power: 175 W, Cycles per burst: 200, Treatment time: 120 sec) using microTUBE with snap cap (Covaris, 520045). After DNA shearing, DNA size selection was performed using AMPure XP beads (Beckman Coulter). Using the size-selected DNA samples, Biotin pull-down and sequencing library preparations were performed using 30 µl of Dynabeads MyOne Streptavidin C1 (Invitrogen, 65001) and a part of Nextera Mate Pair Sample Preparation kit (Illumina, FC-132-1001) following the kit's protocol at half scale. For library amplification, KAPA Real-Time Amplification Kits (KAPA BIOSYSTEMS, KK2701) were used to determine the optimal PCR cycles (9–12 cycles) and the final PCR products were purified by AMPure XP beads. The quality of “Hi-C DNA” NGS libraries were measured by a Bioanalyzer High Sensitivity DNA chip. DNA was ligated with adaptors of Illumina TruSeq Library Preparation Kit (illumina).
GEO Accession
GSM2399022