GSM8403797: NPAT; Homo sapiens; OTHER
Cells were fixed with 1% and 4% formaldehyde HT1080 cells were grown in 6 well plates until 70-80% confluency. After one wash with 1X PBS (GIBCO # 770011-044), cells were crosslinked in 1X PBS with 4% formaldehyde (Thermo Fisher Scientific # 28908) for 10 min at room temperature (RT). In some crosslinking conditions, 1% formaldehyde was used. Crosslink was quenched using 0.25 M Glycine for 5 min at RT. Cells were de-crosslinked overnight at 58°C in 1X PBS. Cytoplasm was extracted for 10-30 min on ice using cytosolic buffer (15mM Tris-HCl, pH7.5, 5mM MgCl2, 60mM KCl, 0.5mM DTT, 15mM NaCl, 300mM sucrose, and 1% NP40) with occasional agitation. After 1X PBS wash, cells were incubated with 1X PBS / 5% BSA / 0.3% Triton™ X-100 for 1 hr at RT with gentle rocking. Primary antibodies were added (1/400 dilution per well) and incubated overnight at 4°C with gentle rocking. After 3 washes with 1X T-PBS (PBS + 0.1% Tween 20) for 5 min at RT, secondary antibodies were added , Alexa Fluor® 488 conjugated goat anti-Mouse IgG (H+L) Superclonal™ or goat anti-Rabbit IgG (H+L) Superclonal™ (corresponding to species of primary antibody; Thermo Fisher # A-28175 and A-27034 respectively) at a dilution of 1:1000 in PBS / 0.3% Triton™ X-100/ 5% BSA for 1 hr at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, cells were incubated for 1 hr at RT with 1 µl of Nt.CviPII-pGL (NEB, 0.5 units) in 800 µl binding buffer per well (20 mM potassium phosphate pH 7.0, 20 mM NaCl, 0.3% Triton™ X-100). In some experiments, heparin (Sigma-Aldrich # H3149) was added at different concentrations to prevent non-specific DNA binding. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer  pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT. Cells were incubated with 10 units of DNA Polymerase I (NEB # M0209S), 5 mM of dNTP mix including Biotin-14-dCTP (Thermo Fisher # 19518018), 5-methyl-dCTP (NEB # N0356S), dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 per well to allow nick translation to occur. Plates were then washed 3 times with washing buffer (20 mM potassium phosphate buffer, pH 7.0, 500 mM NaCl, 0.3% Triton™ X-100) for 15 min at RT with an additional wash with 1X PBS. Cells were then covered with 100 µl of 1X PBS and DNA was extracted using Monarch genomic DNA purification kit (NEB # T3010S) according to manufacturer's recommendations. After DNA purification, 100-500 ng of genomic DNA was fragmented using 2 units of Nt.CviPII (NEB # R0626S) in 500  µl of 1X NEBuffer 2 at 37°C overnight. Nt.CviPII was inactivated at 65°C for 20 min. NaCl 5M stock was added to reach 1 M final concentration along with 30 µl of Streptavidin magnetic beads (NEB # S1420S) for 2 hrs with end-over-end mixing at 4°C to capture biotinylated DNA fragments. Streptavidin bound DNA beads were washed twice with 1X TE buffer supplemented with 2 M NaCl and 0.05 % Triton™ X-100. A third wash was performed with 1X TE buffer. Beads were resuspended in 50 µl of TE buffer and libraries were made on beads using NEBNext®Ultra™ II DNA Library Prep Kit for Illumina® (NEB # E7645S) according to manufacturer's protocol. 8 to 12 PCR cycles were used to amplify barcoded DNA. NEED-seq protocol described above was used with some modifications. Briefly, K562 crosslinked cells with 4% formaldehyde were decrosslinked overnight at 58°C in 1X PBS. Cytoplasmic extraction was performed for 30 min with end-over-end mixing at 4°C. After 300 x g centrifugation at 4°C for 5 min, cell pellet was washed once with 1X PBS and nuclei number was calculated using Luna automated cell counter. Nuclei resuspended in 1X PBS were then captured by adding 10 µl of preactivated Concanavalin A magnetic beads (Cell Signaling Technology # 93569) for 10 min with end-over-end mixing at RT. Bead-bound nuclei were incubated with 1X PBS including 0.3 % Triton X-100 and 5% BSA for 1 hr at RT. Primary antibodies were added (1/400 final dilution) and incubated overnight with end-over-end mixing at 4°C. After 3 washes with 1X T-PBS for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT with gentle rocking. After 3 washes with 1X T-PBS for 5 min at RT, beads were resuspended in 1 ml binding buffer (as described above) including 1 l of fusion enzyme Nt.CviPII-pGL (NEB) for 1 hr at RT. Beads were then washed 3 times with washing buffer (as described above) for 15 min at RT. After 1 wash with 1X PBS, beads were incubated with 10 units of DNA Polymerase I, 5 M of dNTP mix including Biotin-14-dCTP, 5-methyl-dCTP, dATP, dTTP and dGTP for 1 hr at 37°C in 800 µl of 1X NEBuffer 2 with end-over-end mixing. After 3 washes with washing buffer for 15 min at RT, beads were resuspended in 50 mM Tris-Cl, pH 7.5, 1 % SDS, 200 mM NaCl and 2 µl of proteinase K (NEB # P8107S) and incubated overnight at 65°C to lyse nuclei bound to the beads and DNA was extracted using phenol-chloroform and precipitated using glycogen. 200 ng of DNA was fragmented using Nt.CviPII and used to make libraries as described above. For low number of cells, barcoded DNA was amplified using ~18 PCR cycles. To determine the dynamic range of NEED-seq, cells were serially diluted after the labeling reaction. In this modified NEED-seq method, HT1080 cells were grown on 6 well plate to 80% confluency. Cells were incubated for 10 min at 4°C using CSK buffer (10 mM PIPES pH 6.8, 300 mM sucrose, 100 mM NaCl, 1.5 mM MgCl2 and 0.5 % Triton X-100) followed by crosslinking with 4% formaldehyde for 10 min at RT. Glycine was used to quench formaldehyde for 5 min at RT (final concentration 0.25M). After 1X PBS wash, blocking buffer was added to cells for 1h at RT (5% BSA, 1X PBS and 0.3% Triton X-100). Different antibodies (see Table S2) were then added at dilutions recommended by the manufacturer and incubated with cells for 16 hrs at 4°C. After 3 washes with T-PBS (PBS with 0.1 % Tween 20) for 5 min at RT, blocking buffer was added with anti-mouse IgG or anti-rabbit IgG coupled with Alexa 488 for 1h at RT. After 3 washes with T-PBS for 5 min at RT, NEED-seq binding buffer (20 mM KPO4, 150 mM NaCl and 0.3% TritonX-100) was added along with Nt.CviPII-pGL enzyme (1 unit per well) for 1h at 37°C. After 3 washes with NEED-seq washing buffer (20 mM KPO4, 500 mM NaCl and 1% TritonX-100) for 15 min at RT, 1X NEBuffer2.1 was added for 1h at 37°C to allow Nt.CviPII-pGL activity to fragment the targeted DNA. After 1 wash with 1X PBS, genomic DNA extraction was performed using Monarch genomic DNA purification kit (6 wells pooled per antibody) according to NEB recommendations with a minor modification which included a lysis step at 56°C for 1h instead of 10 min to reverse DNA crosslinking. 0.1 to 1 µg of genomic DNA was used for size selection using first 0.4x volume of NEBNext Sample Purification beads with 10 min incubation at RT. Supernatant containing 200-500 bp DNA sizes was separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and collected. An additional 0.4x volume of NEBNext Sample Purification beads was added to the supernatant and incubated for 10 min at RT. After 2 washes with 80% EtOH, DNA was eluted form beads with 50 µl of low TE buffer and DNA libraries were prepared using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on size selected DNA and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. USER enzyme (NEB # E7103L) was incubated for 20 min at 37°C and another round of library size selection was performed as described above. The library was amplified using 13 PCR cycles. PCR products were purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of size selected DNA library was sequenced on Novaseq 6000. HT1080 cells (0.5x10e6) were crosslinked with 4% formaldehyde for 10 min at RT and quenched with 1.5 M Tris-HCl (pH 8) for 5 min at RT. After 1X PBS wash, cells were permeabilized with Hi-C lysis buffer (10 mM Tris-HCl (pH 8), 10 mM NaCl and 0.2% NP40) for 30 min at 4°C, washed with 1X NEBuffer2 and incubated for 10 min at 58°C with 1X NEBuffer2, 0.3% SDS and 1 mM DTT. To quench SDS, Triton X-100 was added (1% final concentration per well) for 15 min at 37°C. 400 U of MboI (NEB # R0147M) were added and incubated for 16 hrs at 37°C. Enzyme was inactivated at 58°C for 20 min. Supernatant was discarded and DNA polymerase I mix containing  NEBuffer2.1 1X (NEB # B7002S), 30 µM of dNTPs (including dCTP/dGTP/dTTP (NEB # N0441S/ N0442S/N0443S respectively) with biotin-14-dATP (ThermoFisher Scientific # 19524016)) and 50 U of DNA polymerase I (NEB # M0210M) was added and incubated for 4 hrs at RT. Ligase mix was added and incubated for 16 hrs at RT. Ligase mix contains 2000 U of T4 DNA ligase (M0202M NEB), 140 µg of recombinant albumin (NEB # B9200S),  1X T4 DNA ligase buffer (NEB # B0202S) and 1 % Triton X-100. After ligation, supernatant was discarded, and cells were washed using 1X PBS. Genomic DNA was extracted from the wells using Monarch Genomic DNA purification Kit (NEB # T3010L). To remove biotin from un-ligated DNA ends, 10 µg of genomic DNA was incubated with 30 U of T4 DNA Polymerase (NEB # M0203L), 25 µM dATP/dGTP (NEB # N0440S/N0442S respectively) in 1X NEBuffer2 for 4 hrs at 20°C. Enzyme was inactivated for 20 min at 75°C. DNA was purified using 1X volume of NEBNext Sample Purification beads (NEB # E6178S) and sheared to 300 bp using Covaris (program : repeat/iterations # 7; repeat process treatment duration 10s; peak power 50W; duty factor 20%; cycles per burst 1000; total treatment time per sample 70s). To capture biotinylated DNA, DNA was incubated with NEB Magnetic Streptavidin beads (NEB # S1420S) for 30 min at RT in 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl. Beads were washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M NaCl and once with low TE buffer. Beads were resuspended in 50 µl of low TE buffer and DNA libraries were prepared on beads using NEBNext Ultra II DNA Library Prep (NEB # E7103L) with some modifications. Briefly, end repair/dA tailing was performed on beads and NEBNext Illumina adaptor (1/10 dilution) were ligated for 2 hrs at RT. 3 µl of USER (NEB # E7103L) was added for 20 min at 37°C. Beads were then washed once with 10 mM Tris-HCl (pH 7.5) supplemented with 1 M of NaCl and once with low TE. Beads were resuspended in 15 µl of low TE. 14 PCR cycles were performed. Supernatant containing PCR products were separated from beads using 12-tube magnetic separation rack (NEB # S1509S) and purified using 0.9x volume of NEBNext Sample Purification beads. 1 nM of DNA Hi-C library was loaded on Novaseq 6000.