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| Status |
Public on Oct 27, 2016 |
| Title |
set7_polyA-mRNA_rep2 |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
genotype/variation: delta set-7
tissue: mycelia
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| Treatment protocol |
[HiC] 2.6 mL of methanol-free formaldehyde (20%) was added to a final concentration of 1%. After a 10 minute room temperature (RT) incubation with shaking at 100 RPM, crosslinking reactions were quenched by adding 3.4 mL of 2 M Tris-HCl [pH 8 @ 25 °C] to a final concentration of 125 mM and incubating at room temperature (RT) for another 10 minutes with shaking at 100 RPM. Conidia were harvested by centrifugation (3000 RPM, 5 minutes) and resuspended gently in 40 mL spheroplasting buffer (1 M sorbitol, 100 mM KPO4 [pH 7.5]) plus 30 mM β-mercaptoethanol, pelleted again (3000 RPM, 5 minutes) and resuspended in 40 mL spheroplasting buffer. 200 mg of VinoTaste Pro (Novozymes) was added and strains were spheroplasted for 60 minutes at 30°C with shaking at 100 RPM. Spheroplasts were harvested by centrifugation (2000 RPM, 5 minutes) and washed 3x with 20 mL HindIII buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.9 @ 25°C], 10 mM MgCl2, 100 μg/mL bovine serum albumin (BSA)). Washed pellets were resuspended in 6 mL HindIII buffer, split into 1 mL aliquots in 1.7 mL eppendorf tubes. Spheroplasts were pelleted (1500 g, 5 minutes) and the supernatant was aspirated away. Aliquots were frozen in liquid nitrogen and stored at -80°C.
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| Growth protocol |
2.5x10^8 conidia were grown in cultures of 50mL 1xVogels, 1.5% Sucrose medium for 3-4 hours at 30oC with shaking at 200rpm (At this time, >70% of the conidia for all strains had germination tubes >4x their original size, as roughly measured by light microscopy)
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| Extracted molecule |
polyA RNA |
| Extraction 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
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
polyA_mRNAseq_libraries
Dataset_S1_4X-changed-genes_WT-v-set7.xlsx
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| Data processing |
Paired-end reads were demultiplexed, trimmed to 50bp (if needed)
Hi-C contact maps were produced according to Imakaev et al. (Imakaev et al. 2012). Briefly, an attempt was made to uniquely map the first 15 base pairs (bp) of each read to the Neurospora genome. If a unique match was not found, the read was extended 5 bp and another attempt was made. This process continued until a unique match was found or the end of the read reached (50 bp).
Read pairs were then assessed using a number of quality control filters, typically removing reads that covered unligated products (Dangling Ends), products that were too close to the restriction site, Self-circles, and extremely large or small fragments.
Read pairs passing all filters (typically 10% of total read pairs) were used to build uncorrected contact maps (arrays) that were then corrected to account for the unequal representation of genomic loci in HiC datasets. Corrected maps were used in all subsequent analyses
To calculate the ratio of observed contacts to expected contacts, the median contact frequency was calculated at each genomic distance (up to a distance of ~3.5 Mb at which point contact frequency had reached background levels) and used as the expected contact frequency at each respective distance.
To directly compare datasets, a fixed number of reads (prior to filtering) were sampled with the program seqtk, such that the sampled number of raw reads from each dataset produced approximately equal numbers of reads that passed the filtering steps.
To make ChIPseq enrichment files across 10kb bins to use for Circos plots, .bcf files were produced using samtools' mpileup function from ChIPseq .bam files, the .bcf files were "fixed" to replace empty positions with zeros, and processed with a custom script to report the ChIP-seq enrichment over 10kb bins (the resulting seven files were catenated into one file).
All python scripts (".py" files) used for Hi-C data processing are available as supplemental information in the manuscript Galazka et al, 2016, Genome Research, except for the modified scripts, plotIntraTelomeres.py and plotInterTelomeres.py, which were used to calculate intra and inter telomeric enrichment in our Hi-C datasets. In addition, the python script K9-K27Quant.py counts the number of Hi-C links originating from a H3K9me3-marked 10kb bin and end in a bin marked by H3K27me2/3, while the python script K9-K27only_Quant.py counts the number of Hi-C links originating from a H3K9me3-marked 10kb bin and end in a bin marked ONLY by H3K27me2/3 (and not H3K9me3). The program count_links_K27marked.py counts the number of Hi-C links in each 10Kb bin marked by H3K27me2/3. The program hicEigenvectorAnalysis.py derives the number of principle components for the whole genome specified using non-iteratively corrected Hi-C .hdf5 datasets. The program hicEigenvector_singleLG.py extracts the Eigenvector of a single Linkage Group from the whole genome data derived from the "hicEigenvectorAnalysis.py" script. The program hicPearsonCorrelationHeatmap.py calculates the Pearson Correlation between all genomic regions and displays them as a heatmap for a specific Linkage Group. These scripts are provided here.
To determine differences in the genome organization caused by mutants, mutants were compared to the WT Hi-C data (NMF39 [GSM1825702] within the GEO dataset GSE71024). See GSM2186732 in this GSE82222 dataset.
To make ChIPseq enrichment files for display in Integrative Genomics Viewer (IGV, Robinson et al, 2011, Nat. Biotechnol.), .bam files (and the corresponding .bai index files) were counted with the "Count" command of igvtools, using the mean window function and 200bp windows. Output file was a .tdf file, displayable on IGV.
For Poly-A mRNA seq, high quality, adapter-less reads were identified (Stacks); Kmer filtering reduced sequencing errors. Reads were mapped (TopHat) to the modified Neurospora genome assembly 12, and sorted (SAMTools), and directionality-preserved read numbers were calculated at each gene ID (HTSeq), adding one read count to prevent abnormal fold changes. Differential gene expression analysis (DESeq) examined replicate differences and pair-wise analyses between WT and mutants. Output .bed files of genes with log2 ≥ 2 or log2 ≤ -2 expression and adjusted p-values ≤ 0.05 were modified as .txt files for Circos plot display.
Genome_build: Neurospora crassa assembly 12 Fixed (files: neurospora_crassa_or74a_12_genome_FIXED.fasta, and neurospora_crassa_or74a_12_transcripts_FIXED.gtf)
Supplementary_files_format_and_content: for Hi-C files: "...array.txt" files are the generated tab-delininated files with raw counts (on a log2 scale) for each bin (the entire genome is divided into 40kb, 50kb, or 10kb segments, and a bin indicates the intersection of two of these segments in an array format) across the genome. "...observed_expected.txt" is a file where the above array file containing observed interactions in our HiC dataset was normalized by the "expected" interaction if interactions were locally strong and stochastically decreased as genomic distance increased.
Supplementary_files_format_and_content: For ChIP-seq files: The tdf files were generated using the "count" function in igvtools (Integrative Genomics Viewer; Broad Institute, Robinson et al, 2011), using a window size of 200bp (.tdf files are binary files that shows enrichment peaks for each ChIP sample and have been processed for faster display of the data in IGV). The bcf files were produced from bam files (mapped to the Neurospora crassa assembly 12 Fixed by samtools), and report enrichment values.
Supplementary_files_format_and_content: For RNA-seq files: the .txt files were generated by the program HTseq (using sequencing reads aligned to the Neurospora crassa assembly 12 Fixed genome with the program samtools). These HTseq.txt files report read number counts per gene for each replicate sample (two replicates total) from our four strains (WT N3752, delta set-7 N4718 and N4730, delta npf N4721, delta set-7;delta dim-5 N5916). The three Dataset.xls files were produced using the replicate HTseq.txt files in the program DESeq to determine genes that were differentially expressed > four fold and were significant (p < 0.05) in each mutant strain when compared to the WT N3752 reference expression datasets.
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| Submission date |
Jun 03, 2016 |
| Last update date |
May 15, 2019 |
| 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 |
GPL20660 |
| Series (1) |
| GSE82222 |
Neurospora crassa genome organization requires subtelomeric facultative heterochromatin |
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| Relations |
| Reanalyzed by |
GSE128317 |
| BioSample |
SAMN05201327 |
| SRA |
SRX1818763 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2186743_ACTGAT_N4730_set7_rep2_polyA-mRNA_HTseq-genecounts.txt.gz |
38.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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