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| Status |
Public on Dec 29, 2017 |
| Title |
N5862_OY350_FGSC4641_polyA-mRNA |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
tissue: germinated conidia
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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 |
total RNA |
| Extraction protocol |
RNA-seq: Mycelia were harvested and added to a tube containing ~350uL glass beads (260-300 micrometer, acid washed [Sigma Aldrich] and resuspended in sdH2O), 350uL NETS buffer (10mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 0.2% SDS) and 350uL phenol:chloroform:isoamyl alcohol (25:24:1) and lysed by a bead beater for 3 minutes at room temperature. Samples were cleared by centrifugation, and the aqueous (top) phase was mixed with 350uL of chloroform. After centrifugation, the aqueous phase was divided into two tubes containing 650uL cold 100% EtOH. Samples were precipitated at -20oC, pelleted by centrifugation, washed twice with 1mL 70% EtOH, and resuspended in DEPC-treated dH2O. RNA was treated with DNase (DNA-free™ DNA Removal Kit, Thermo Fisher Scientific) and cleaned (Agencourt® RNAclean XP® beads, Beckman Coulter).
ChIP-seq: 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 2uL) 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.
Whole-genome-seq: Used input DNA from ChIP experiments.
RNA-seq: RNA-seq libraries were prepared (KAPA Stranded mRNA-seq kit, KAPA Biosystems).
ChIP-seq: 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, 10 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.
Whole-genome-seq: Performed library prep same as ChIP-seq samples.
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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_library
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| Data processing |
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 translocations.
To make ChIPseq enrichment files for display in Integrative Genomics Viewer (IGV, Robinson et al., 2011, Nat. Biotechnol.), reads were mapped with bowtie2 to the nc12-fixed genome (Galazka et al., 2016) using default parameters, and reads were converted to .bam files, sorted, and indexed. The .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 whole-genome-seq breakpoint analysis: Incongruous-paired and split-read alignments for input DNA samples were found using bwa-mem. The discordant and split-read alignments were analyzed with LUMPY v0.2.9 to determine the location of chromosomal breakpoints (Layer et al. 2014). A list of predicted breakpoints with a minimum call weight of five was generated and calls with an evidence set score <0.05.
Genome_build: Neurospora crassa assembly 12 Fixed (files: neurospora_crassa_or74a_12_genome_FIXED.fasta, and neurospora_crassa_or74a_12_transcripts_FIXED.gtf; files are found in GEO submission GSE71024).
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 25 -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 Poly-A mRNA files: Text files contain counts for every gene as outlined in process above.
Supplementary_files_format_and_content: For whole-genome-seq: bedpe files contain list of predicted breakpoints predicted by LUMPY.
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| Submission date |
Sep 19, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Eric Selker |
| E-mail(s) |
selker@uoregon.edu
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| Organization name |
University of Oregon
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| Department |
Biology
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| Street address |
1370 Franklin Blvd.
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| City |
Eugene |
| State/province |
Oregon |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL20660 |
| Series (1) |
| GSE104019 |
Telomere repeats induce domains of H3K27 methylation in Neurospora |
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| Relations |
| BioSample |
SAMN07671965 |
| SRA |
SRX3198482 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2787835_GTGGCC_N5862-rep1_polyA-RNA_counts.txt.gz |
38.6 Kb |
(ftp)(http) |
TXT |
| GSM2787835_GTTTCG_N5862-rep2_polyA-RNA_counts.txt.gz |
38.6 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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