 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 19, 2016 |
| Title |
N5580_hpo_HiC |
| Sample type |
SRA |
| |
|
| Source name |
germinated condia
|
| Organism |
Neurospora crassa |
| Characteristics |
genotype: N5580 hpo
|
| Treatment protocol |
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.
|
| 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)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
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.
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.
Indexed paired-end HiC libraries were pooled and sequenced either on an Illumina HiSeq 2500 as either 100nt or 50nt paired end sequencing runs, or a NextSeq500 as a 75nt paired-end sequencing run at the University of Oregon Genomics Core Facility
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
genomic DNA HiC libraires
|
| Data processing |
Paired-end reads were demultiplexed, trimmed to 50bp (if needed)
HiC 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 ChIP-seq enrichment files across 10kb bins to use for Circos plots, .bcf files were produced using samtools' mpileup function from ChIP-seq .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).
Genome_build: Neurospora crassa assembly 12, but with the large inversion on LGVI (as evident from our data) corrected. (files: neurospora_crassa_or74a_12_genome_FIXED.fasta, and neurospora_crassa_or74a_12_transcripts_FIXED.gtf)
Supplementary_files_format_and_content: "...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 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.
|
| |
|
| Submission date |
Jul 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew David Klocko |
| E-mail(s) |
aklocko@uccs.edu
|
| Phone |
719-255-3255
|
| Organization name |
University of Colorado Colorado Springs
|
| Department |
Chemistry and Biochemistry
|
| Lab |
Klocko
|
| Street address |
278 Centennial Hall, 1420 Austin Bluffs Pkwy
|
| City |
Colorado Springs |
| State/province |
Colorado |
| ZIP/Postal code |
80918 |
| Country |
USA |
| |
|
| Platform ID |
GPL20705 |
| Series (1) |
| GSE71024 |
HiC of Wild Type Neurospora crassa and mutants deficient in heterochromatin formation |
|
| Relations |
| BioSample |
SAMN03863735 |
| SRA |
SRX1099809 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1825704_N5580_hpo_49MreadsSAMPLED_wholegenome_HiCarray_10k_observed_expected.txt.gz |
46.9 Mb |
(ftp)(http) |
TXT |
| GSM1825704_N5580_hpo_wholegenome_10kb-res_HiC_array.txt.gz |
83.9 Mb |
(ftp)(http) |
TXT |
| GSM1825704_N5580_hpo_wholegenome_50kb-res_HiC_array.txt.gz |
6.7 Mb |
(ftp)(http) |
TXT |
| GSM1825704_N5580_hpo_wholegenome_HiCarray_10k_observed_expected.txt.gz |
47.3 Mb |
(ftp)(http) |
TXT |
| GSM1825704_N5580_hpo_wholegenome_HiCarray_50k_observed_expected.txt.gz |
3.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
