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| Status |
Public on Jun 10, 2024 |
| Title |
NKL2_LGIIK9het25-deletion_H3K9me3_rep2 |
| Sample type |
SRA |
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| Source name |
mycelia
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| Organism |
Neurospora crassa |
| Characteristics |
tissue: mycelia
cell type: asexual fungal hyphae
genotype: mat A; {delta}LGIIK9het25::hph
treatment: overnight growth (16hrs) in 1xVogels+1.5% sucrose
chip-seq antibody: Active Motif anti-H3K9me3 (cat# 39161)
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| Treatment protocol |
Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 15 min in 0.5% formaldehyde at room temperature, with shaking at 100 rpm, and then quenched with 0.125M glycine for histone modification ChIP.
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| Growth protocol |
Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% triton + proteinase inhibitors) and was disrupted by sonication, which simultaneously sheared chromatin, using a Bioruptor Pico (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 nutated overnight at 4oC. The next day, equilibrated protein A/G magnetic agarose beads (Pierce/Thermo Scientific; cat # PI78609) was added to bind the antibody, incubated for 2 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 RNaseA treated for two hours at 50oC, then proteinase K treated for 2 hours at 50oC, the DNA was extracted with phenol:chloroform:IAA (25:24:1), and DNA was precipitated with ethanol, sodium acetate, and glycogen, and allowed to precipitate overnight at -80oC. The next day, samples were precipitated by centrifugation, washed, dried in a Speedvac and resuspended in 30uL TE. ChIP DNA was quantified with a Qubit 3.0 using the HS method.
Approximately 10 ng of DNA was used to generate ChIP-Seq libraries. Each library was prepared using the NEB Next Ultra II Library Preparation Kit (New England Biolabs, #E7645L) with the adapters from the NEBNext® Multiplex Oligos for Illumina®(Index Primers Set 1 [E7335L] or 2 [E7500L]) according to the manufacturer’s instructions, except that final libraries were only PCR-amplified using eight cycles for amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw ChIP-seq data files, as fastq files, were mapped to version 14 of the Neurospora crassa genome (nc14) [Rodriguez et al., 2022 [Pubmed ID: ] with bowtie2 (version 2.4.2), and output sam files were converted to sorted bam files using samtools (version 1.14), which were used by Deeptools (version 3.5.0) to produce bigwig files with 25 basepair bin resolution, normalized by Reads per Kilobase Per Million Reads (RPKM), for display on the Integrative Genomics Viewer (IGV)
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 bigwig files are generated by Deeptools; briefly, these files divide the genome into bins and the number (count) of sequencing reads per bin is reported for each bin; the bigwig file format is a single column with the enrichment per bin displayed, and the seven headers indicate the chromosome and bin size.
Supplementary files format and content: the bedgraph files are generated by Deeptools; briefly, these files divide the genome into bins and the number (count) of sequencing reads per bin is reported for each bin; the bedgraph file format is a four column file, with the first column being the chromosome, second column being the bin start, third column being the bin stop, and fourth column being the bin enrichment
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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 |
GPL26551 |
| Series (2) |
| GSE269228 |
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa [ChIP-Seq] |
| GSE269235 |
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa |
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| Relations |
| BioSample |
SAMN41708027 |
| SRA |
SRX24823089 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8309020_NKL2_deltaLGIIK9het25_rep2_H3K9me3_ChIPseq-nc14_50bp_normRPKM.bigwig |
4.4 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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