title: GSM2186735: N5542_set7dim5_HiC; Neurospora crassa; OTHER
library_construction_protocol: [HiC] To determine the concentration of genomic DNA (gDNA) per aliquot of spheroplasts, 1 aliquot per replicate was resuspended in 200 μL decrosslinking buffer (50 mM Tris-HCl [pH 8], 5 mM Na Ethylenediaminetetraacetic acid (Na-EDTA), 0.5% (w/v) sodium dodecyl sulfate (SDS)) plus 100 mg proteinase K and incubated at 65 °C overnight. The next day, the volume was brought to 500 uL with TE buffer (10 mM Tris-HCl [pH 8], 1 mM Na-EDTA) plus 40 mg RNase A and aliquots were incubated at 37°C for 30 minutes. gDNA was then extracted twice with a 1:1 mixture of buffered phenol (pH 8) and chloroform, and once with chloroform. gDNA was precipitated by adding 1/10 volume of Na acetate (pH 5.2) and 1 volume of isopropanol. DNA pellets were washed 3x with 70% (v/v) ethanol, dried at RT for 10 minutes and resuspended in 50 μL TE. The DNA concentration was determined from the absorbance at 260 nm. Spheroplasts corresponding to 3.5 μg of gDNA were resuspended in 270 μL of HindIII buffer and 30 μL of 10% (w/v) SDS was added to a final concentration of 0.1% (w/v). Spheroplasts were then lightly decrosslinked by incubation at 65°C for 10 minutes and then placed immediately on ice. 33 μL of 10% (v/v) Triton X-100 and 8.3 μL of 10x HindIII buffer was added and gDNA digested with 400 U (20 μL of 20 U/μL from NEB) HindIII for 16 hours at 37 °C while nutating. The next day, samples were placed on ice and split into two aliquots. To construct HiC libraries, one aliquot of HindIII digested chromatin was labeled with biotin-14-dCTP in a 210 μL reaction consisting of 30 μM dATP, 30 μM dTTP, 30 μM dGTP, 30 μM biotin-14-dCTP and 25 U of Klenow (large fragment). This reaction was incubated at 37 °C for 45 minutes with nutating. Klenow (large fragment) and HindIII was then inactivated by the addition of SDS to 1.5% (w/v) and incubation at 65°C for 30 minutes. This chromatin was then used in a 3.5 mL ligation reaction consisting of 50 mM Tris-HCl [pH 7.5 @ 25 °C], 10 mM MgCl2, 0.9% (v/v) Triton X-100, 10 mM Dithiothrireitol (DTT), 100 μg/mL BSA, 1 mM adenosine triphosphate (ATP) and 1675 U T4 DNA ligase (4.2 μL of 400,000 U/mL T4 DNA ligase from NEB). This reaction was incubated at 16°C for 4 hours. At the end of the 4 hour 3C and HiC ligation reactions, 250 mg of proteinase K was added and all reactions were allowed to decrosslink at 65°C overnight. The next day, another 250 mg of proteinase K was added and samples were decrosslinked at 65°C for another 2 hours. Samples were cooled to ~25°C on ice, and extracted with 5 mL of phenol (pH 8) by vortexing for 2 minutes. After centrifugation (3500 RPM, 10 minutes), the aqueous phase was removed and extracted with 5 mL of phenol (pH 8):chloroform (1:1) by vortexing for 2 min. After centrifugation (3500 RPM, 10 minutes), the aqueous phase was removed and brought to a volume of 5 mL with TE. DNA was precipitated by the addition of 0.5 mL 3 M Na Acetate [pH 5.2], 50 mg glycogen, 12.5 mL 95% ethanol and incubation at -80°C for 1 hour. DNA was pelleted by centrifugation (20,000 g, 20 minutes, 4°C), the supernatant decanted and DNA resuspended in 450 μL TE by vortexing. DNA was again extracted with 1 volume of phenol (pH 8):chloroform (1:1) and then chloroform as before. DNA was then ethanol precipitated as before and resuspended in 25 μL of TE/10 buffer (10 mM Tris-HCl [pH 8 @ 25°C], 0.1 mM Na-EDTA). To remove biotin-dCTP from unligated ends, the HiC libraries were digested for 2 hours at 12°C in a 50 μL reaction consisting of 5 μL of NEB buffer 2, 100 μg/mL BSA, 100 μM dATP, 100 μM dGTP and 5 U of T4 DNA polymerase. These reactions were quenched by adding Na-EDTA to 10 mM and DNA extracted with 1 volume of phenol (pH 8):chloroform and then with 1 volume of chloroform. DNA was ethanol precipitated, resuspeneded in 500 μL of TE and sheared by sonication (duty cycle 80, power 1.2, 10 s, 5x with 1 minute rest on ice between pulses). Sheared, biotinylated DNA was captured by adding 25 μL of streptavidin beads (Invitrogen) that had been equilibrated in BW buffer (5 mM Tris-HCl [pH 8 @ 25°C], 0.5 mM Na-EDTA, 1 M NaCl, 0.05% (v/v) Tween-20) and incubating for 30 minutes with shaking. Beads were washed 2x with BW buffer, 2x with TE/10 buffer and resuspended in 25 μL TE/10 buffer. [HiC] To prepare HiC libraries for Illumina sequencing, either Illumina TruSeq kits was used according to the manufacturers’ protocol, or essentially a “homemade version” of that protocol was used. In the homemade version, DNA ends were repaired in reactions consisting of 25 μL of HiC libraries on streptavidin beads, 5 μL of T4 DNA ligase buffer, 2 μL 10 mM (each) dNTPs, 0.5 μL end repair enzyme mix (0.5 U T4 DNA polymerase, 0.25 U Klenow Fragment, 2.25 U T4 DNA Polynucleotide Kinase) in a final volume of 50 μL and incubated at 20°C for 30 minutes. Beads were washes 2x with BW buffer and 2x with TE/10, resuspended in 16.5 μl TE/10. A-tailing was performed by adding 2 ul 10x NEB Buffer #2, 1 ul 4 mM dATP, 0.5 ul Klenow 3’ to 5’ exo minus (5 U/ul) and incubating at 37 C for 30 minutes, mixing every 10 minutes. Beads were again washed with BW and TE/10 buffers as above and resuspended in 20 ul TE/10. Indexed adaptors were ligated by adding 25 ul 2x ligase buffer (132 mM Tris-HCl [pH 7.6], 20 mM MgCl2, 2 mM DTT, 2 mM ATP, 15% (w/v) PEG 6000), 2.5 ul T4 DNA ligase (400 U/ul) and 2.5 ul Illumina TruSeq DNA adapters diluted 1:10 and incubating at RT for 20 minutes, mixing every 10 minutes. Ligation reactions were quenched with 5 ul of 0.5 M Na-EDTA [pH 8] and beads washed 3x with BW buffer and 2x with TE/10. Beads were then resuspended in 50 ul TE/10 and HiC libraries amplified in 50 ul PCR reactions using the Phusion polymerase and the following cycle: 45 s at 98°C; 18 cycles of 15 s at 98°C, 30 s at 63°C, 30 s at 72°C; 1 minute at 72°C. The PCR product was separated from the beads, cleaned with Ampure XP beads, and resuspended in 25 ul TE/10. Library quality was assessed by gel electrophoresis before deeps sequencing. [ChIP] Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 10 min in 0.5% formaldehyde for histone modification ChIP.  Tissue added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% triton + proteinase inhibitors) and was disrupted by sonication for thirty pulses before chromatin was sheared using a Bioruptor (Diagenode) for 15 min with a cycle of 30 sec on followed by 30 sec off, at high power. Lysates were cleared by centrifugation, 1/20th Input was saved, and histone modification antibody was added (typically 1uL) and samples were rotated overnight at 4oC. The next day, equilibrated Protein A or Protein G slurry (agarose or magnetic beads) was added to bind the antibody, incubated for 3 hours at 4oC, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl was buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40), and DNA/protein was eluted with TES buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) and incubated at 65oC.  Samples (IP and input) were decrosslinked at 65oC overnight.  The next day, samples were proteinase K treated for 2 hours at 50oC, and DNA was purified using the QIAquick PCR purification kit  and eluted in 30 μl of water.  Approximately 10 ng of DNA was used to generate ChIP-Seq libraries.  Each library was prepared using the Illumina Tru-Seq kits A and B (Illumina, IP-202-1012 and IP-202-1024) according to the manufacturer’s instructions.  “Invisible” fragments between 250-400 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606).  Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 8 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min. [RNA] Cultures were grown in Vogels m