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| Status |
Public on Jun 10, 2024 |
| Title |
N4933_delta-K27-region_DpnII-insitu-HiC_rep1 |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
tissue: germinated conidia
cell type: hyphae
genotype: mat a; {delta}K27region (delta 47.4kb)::hph
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| Treatment protocol |
Methanol-free formaldehyde was added to a final concentration of 1% and cultures were incubated 10 minutes at room temperature (RT; ~25°C) with shaking at 100 RPM for crosslinking; 1 M Tris-HCl [pH 8 at 25°C] (Hoffman et al. 2015) was added to a final concentration of 125 mM and cultures were incubated at RT for 10 minutes, shaking at 100 RPM, to quench the reaction. Conidia were harvested by centrifugation (4000 RPM, 3 min) and resuspended gently in 20 mL spheroplasting buffer (1 M sorbitol, 100 mM KPO4 [pH 7.5]) with b-mercaptoethanol added to 30mM. Conidia were pelleted again and resuspended in 20 mL spheroplasting buffer with b-mercaptoethanol added to 1mM. VinoTaste (Novozymes; powder dissolved in water to a stock solution of 50 mg/mL) was added to 50mg and strains were digested at 30°C, shaking at 100 RPM, for 60 min. Spheroplasts were harvested by centrifugation (3500 RPM, 2 min) and pellets were washed twice with 20 mL Spheroplasting buffer, followed by three washes of the buffer of the 1x restriction enzyme to be used for digestion, here DpnII buffer (100 mM NaCl, 50 mM Tris-HCl [pH 7.9 at 25°C], 10 mM MgCl2, 100 μg/mL BSA). Washed pellets were resuspended in 3.5 mL of the same restriction enzyme buffer, split into four – 1 mL aliquots in 1.7 mL Eppendorf tubes, pelleted (10,000 rpm, 5 min), whereupon the supernatant was removed, and spheroplast pellets were stored at -80°C.
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| Growth protocol |
To grow WT Neurospora culture, 25 mL cultures containing 1x Vogel’s medium + 1.5% sucrose were inoculated with 1.25x10^8 conidia, (harvested from ~six baby slants by vortexing with water; conidia were washed twice prior to use) and grown at 32 °C with shaking at 200 revolutions per minute (RPM) for ~four hours until ~90% of conidia had germinated, and growth tubes were ~4x the length of the initial conidia.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
processed matrices contain genomic loci in defined bin sizes for the column names
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| Data processing |
All in situ Hi-C contact maps and other analyses presented in this manuscript were generated using the program suite HiCExplorer (https://hicexplorer.readthedocs.io/en/latest/index.html) (Ramírez et al. 2018), using version 3.5 for all analyses. Briefly, raw fastq files were mapped to the Neurospora genome (version 14 [Rodriguez et al., 2022 [Pubmed ID: 35244156]) using bowtie2 (version 2.3), with the --local and --reorder flags, and the Hi-C contact matrix was built with hicBuildMatrix using the default settings (with both DpnII [GATC] and MseI [TTAA] restriction sites used for quality control), and resulting images of the contact matrix images were made with hicPlotMatrix; (total) merged matrices were constructed by first merging all replicates using the cat command and processing them in an identical manner. Contact map images are either presented as raw or Knight-Ruiz corrected (Knight and Ruiz 2012), performed with the hicCorrectMatrix program, to account for differences in restriction enzyme site distribution or sequencing bias of underlying DNA. Differences between wild type datasets (from Rodriguez et al., 2022 [Pubmed ID: 35244156]) and mutant datasets were performed with hicCompareMatrices in which the experimental (mutant) matrix was listed first and the control (wild type) matrix was listed second; all compare matrices used the --log2diff flag to obtain the log2 difference in contact strength. Wild type matrices with approximately the same number of valid reads as the mutant dataset were generated by selecting the appropriate number of total reads from fastq files of the wild type datasets (Rodriguez et al., 2022) using the sed command, reads were mapped to the nc14 genome, and the contact matrix built with hicBuildMatrix as above.
Assembly: Assembly: nc14_genome-seq-name.fasta (version 14; Rodriguez et al., [Pubmed ID: 35244156]), or the NKL2 genome generated here (deleted of the LGIIK9het25 constitutiive heterochromatic region).
Supplementary files format and content: the .h5 files are contact matrices generated by hicExplorer; briefly, this 3D matrix is divided into bins, and the interactions between the bins is reported - either raw contact counts, Knight-Ruiz corrected contact counts, or the calculated log2 difference in contacts between a wild type strain (wild type data from Rodriguez et al., 2022 [Pubmed ID: 35244156]) and the mutant strains presented here.
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| Submission date |
Jun 06, 2024 |
| Last update date |
Jun 10, 2024 |
| Contact name |
Andrew David Klocko |
| E-mail(s) |
aklocko@uccs.edu
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| Phone |
719-255-3255
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| Organization name |
University of Colorado Colorado Springs
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| Department |
Chemistry and Biochemistry
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| Lab |
Klocko
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| Street address |
278 Centennial Hall, 1420 Austin Bluffs Pkwy
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| City |
Colorado Springs |
| State/province |
Colorado |
| ZIP/Postal code |
80918 |
| Country |
USA |
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| Platform ID |
GPL20705 |
| Series (2) |
| GSE269229 |
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa [Hi-C] |
| GSE269235 |
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa |
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| Relations |
| BioSample |
SAMN41706991 |
| SRA |
SRX24822646 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8309022_N4933-delta-K27-region_DpnII_rep1-022019_nc14_1kb-matrix.h5 |
4.0 Mb |
(ftp)(http) |
H5 |
| GSM8309022_N4933-delta-K27-region_DpnII_rep1-022019_nc14_20kb-matrix.h5 |
1.3 Mb |
(ftp)(http) |
H5 |
| GSM8309022_N4933-delta-K27-region_DpnII_rep1-022019_nc14_40kb-matrix.h5 |
606.2 Kb |
(ftp)(http) |
H5 |
| GSM8309022_N4933-delta-K27region_nc14-1kb-matrix.h5 |
7.6 Mb |
(ftp)(http) |
H5 |
| GSM8309022_N4933-delta-K27region_nc14-20kb-matrix-KR.h5 |
10.4 Mb |
(ftp)(http) |
H5 |
| GSM8309022_N4933-delta-K27region_nc14-20kb-matrix.h5 |
1.8 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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