GSM8309022: N4933_delta-K27-region_DpnII-insitu-HiC_rep1; Neurospora crassa; Hi-C
First, the concentration of genomic DNA per spheroplast aliquot was determined by resuspending one aliquot per culture replicate in 200 μL decrosslinking buffer (50 mM Tris-HCl [pH 8], 5 mM sodium ethylenediaminetetraacetic acid [Na EDTA], 0.5% [w/v] sodium dodecyl sulfate [SDS]) plus 100 mg proteinase K (Invitrogen) and incubated at 65°C for 16 hours. The volume was then increased to a total of 500 μL using TE buffer (10 mM Tris-HCl [pH 8], 1 mM Na-EDTA), and 40 mg RNase A (Invitrogen) was added. Aliquots were incubated at 37 °C for 30 min, and genomic DNA was extracted with twice 25:24:1 phenol:chloroform:isoamyl alcohol (25:24:1; ThermoFisher cat# AC327115000), once with chloroform, and precipitated with 0.1 volume sodium acetate (pH 5.2) and 1 volume isopropanol, centrifuged (10 minutes, 13k rpm), and the resulting DNA pellet was washed once with 70% (v/v) ethanol, dried for 10 min in a Savant SpeedVac (ThermoFisher, Model # DNA120-115), and resuspended in 50 μL TE. The total DNA concentration per pellet was determined by quantifying 2 μL of the total resuspension of genomic DNA (50 μL) using a Qubit 3.0 HS DNA quantification kit (ThermoFisher cat# Q33217). Spheroplasts containing 3.5 μg of genomic DNA were resuspended in 270 μL of 1x restriction enzyme buffer (either DpnII, above) and 50 μL hydrated glass beads were added (Sigma-Aldrich #G1145, Acid washed, 150-212 μm). Spheroplasts were lysed by vortexing tubes for 30 seconds followed by 30 seconds on ice; five total cycles were performed. Following the settling of the beads, 270 μL of supernatant (and cell debris, if possible) were moved to a new tube. SDS was added to 0.625% final concentration (30 μL of 6.25% stock), tubes incubated at 62 °C for 7 minutes, and immediately placed on ice. Triton X-100 was added to a final concentration of 1% [33 μL of 10% (v/v)] and 10x DpnII or MseI buffer was added to account for added volumes of SDS, Triton and enzymes. Genomic DNA in chromatin was then digested with 200 U DpnII and MseI (double digest) for 16 hours at 37°C while nutating. Samples were then centrifuged for 3000 rpm for 10 minutes, the supernatant was removed, and the pellets, which contained permeabilized nuclei and chromatin, were resuspended in 83 μL of 1x DpnII buffer. Sticky ends were blunted in a 100 μL reaction with 30 μM each of dCTP, dGTP, dTTP, Biotin-14-dATP (Invitrogen cat# 19524-016), and 25 U of Klenow (large fragment). Reactions were mixed by pipetting and incubated at 37°C for 60 minutes with nutating. An additional 110 μL of 1x DpnII or MseI buffer was added to the samples (210 μL total chromatin volume), and ligation reactions with 1x T4 Ligation buffer, 1% Triton X-100, 100 μg/mL BSA, and 1675 U of T4 DNA ligase were immediately prepared and incubated at 16 °C for 4 hours. Proteinase K (250 μg total; Gold Biotechnology cat# 97062-238) was added and reactions were decrosslinked at 65°C once overnight (~16 hours) and following the addition of another 250 μg of proteinase K, reactions were decrosslinked a second time for 2 hours. Cooled samples were extracted twice with phenol:chloroform:isoamyl alcohol, the aqueous fraction was transferred to a 2.0 mL microcentrifuge tube, and the volume increased to 500 μL with additional TE buffer. Ligated DNA circles were precipitated by addition of 0.1x volume 3M sodium acetate pH 6.0, 2.5x volumes 100% ethanol, 40 μg glycogen (ThermoFisher cat# R0561), incubation at -80 °C for 30 minutes followed by centrifugation at 13,000 rpm for 15 minutes. Pellets were washed once with 70% ethanol, dried at RT for 10 minutes in a speedvac, and resuspended in 50 μL TE. RNaseA (20 μg; SigmaAldrich cat# R4875) was added and samples were incubated at 37 °C for 30 minutes. An additional 450 μL TE was added, and ligation products were extracted once with 25:24:1 phenol:chloroform:isoamyl alcohol and once with chloroform. The aqueous fraction was transferred to a new 2.0 mL microcentrifuge tube, and ligation products were precipitated (0.1x 3M sodium acetate pH 6.0 and 2.5x 100% ethanol were added, and tubes were incubated at -80 °C for 20 minutes and centrifuged at 13,000 rpm for 10 minutes), washed, dried, resuspended in 25 μL TE/10 (10 mM Tris-HCl [pH 8 at 25 °C], 0.1 mM sodium EDTA) and stored at -20 °C. To remove biotin-dATP from unligated DNA ends, 50 μL reactions consisting of 1x NEB buffer 2.1, 100 μM each of dATP and dGTP, and 5 U of T4 DNA polymerase were incubated at 12 °C for 2 hours and quenched with EDTA (final concentration 10 mM), and the reaction volume was increased to 500 μL with TE/10. DNA ligation products were extracted once with 25:24:1 phenol:chloroform:isoamyl alcohol and once with chloroform, transferred to a 2.0 mL microcentrifuge tube, and ethanol precipitated (0.1x volume 3M sodium acetate, pH 6.0 and 2.5x volume 100% ethanol were added, tubes were incubated at -80 °C for 20 minutes, centrifuged at 13,000 rpm for 10 minutes, washed, dried, and DNA pellets were resuspended in 500 μL TE/10). Ligation products were sheared by sonication (Qsonica, Model Q55; amplitude = 30%, 30 seconds sonication, 30 second incubation on ice between pulses). Sheared, biotinylated ligation products were captured on streptavidin magnetic beads (Dynabeads M280, Invitrogen cat# 11205D) equilibrated with 1x BW buffer (5 mM Tris-HCl [pH 8 at 25°C], 0.5 mM Na-EDTA, 1 M NaCl, 0.05% [v/v] Tween-20) at RT for 30 minutes, with pipetting every 10 minutes. Beads were washed on a magnetic rack three times with 1x BW buffer, twice with TE/10, and resuspended in 25 uL TE/10 buffer. Hi-C libraries for Illumina sequencing were prepared using a NEBNext Ultra II kit (New England Biolabs cat# E7645) with corresponding Multiplex Barcode sets (NEB cat# E7335 and E7500) according to the manufacturer's protocol, with the following exceptions, as all steps were performed using the Hi-C library attached to the magnetic streptavidin beads: following the adapter ligation and USER enzyme digestion, magnetic beads were washed five times with 1x BW buffer (5 mM Tris-HCl [pH 8 at 25°C], 0.5 mM Na-EDTA, 1 M NaCl, 0.05% [v/v] Tween-20) and once with TE/10, and resuspended in 15 μL TE/10 (10 mM Tris-HCl [pH 8 at 25 °C], 0.1 mM sodium EDTA); PCR enrichment of the barcoded Hi-C library off streptavidin beads used eight and following the separation of the aqueous PCR product from the magnetic beads, the PCR product was cleaned with a 1:1 ratio of Ampure XP beads (Agencourt, Beckman-Coulter) per the manufacturer's protocol, resuspended in 25 μL TE/10, and quantified by a Qubit HS reaction. Prior to sequencing, all libraries were assessed for quality via Fragment Analyzer and qPCR (Genomics and Cell Characterization Core Facility [GC3F], University of Oregon). Indexed in situ Hi-C libraries were pooled and sequenced on an Illumina HiSeq 2500 at the Genomics and Cell Characterization Core Facility [GC3F] at the University of Oregon as 100 nucleotide (nt) paired-end (PE100) sequencing runs.