 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 16, 2019 |
| Title |
N5801_dim1-2_polyA_RNA |
| Sample type |
SRA |
| |
|
| Source name |
germinated conidia
|
| Organism |
Neurospora crassa |
| Characteristics |
tissue: germinated conidia
|
| Treatment protocol |
Frozen cultures were ground in a mortar and pestle kept cold with LN2, and resuspended with an equal volume (weight:volume) of Buffer A (1M sorbitol, 7% Ficoll, 20% Glycerol, 5mM Magnesium Acetate [MgAc2], 3mM CaCl2, 50mM Tris HCl pH7.5). Culture slurry was filtered through a cheesecloth funnel prewet with Buffer A. Buffer B (10% Glycerol, 5mM MgAc2, 25mM Tris-HCl pH7.5) was added (2x the volume of buffer A) to the supernatant. Following overlaying of the solution on additional Buffer A:Buffer B solution (ratio 2.5:4), the supernatant containing cytoplasm and nuclei was centrifuged at 3000xg for 7 minutes in an HB-4 swinging bucket rotor at 4oC to pellet cell debris. Supernatant was overlaid onto ~1/7 total volume Buffer D (1M Sucrose, 10% Glycerol, 5mM MgAc2, 25mM Tris-HCl pH7.5), and centrifuged at 9400xg for 15 minutes in an HB-4 rotor. Supernatant was discarded and the pelleted nuclei were resuspended in Nuclei Storage Buffer (25% Glycerol, 5mM MgAc2, 0.1mM EDTA, 3mM DTT, 25mM Tris-HCl pH 7.5), flash frozen in LN2, and stored at -80°C for use in subsequent experiments.
|
| Growth protocol |
Conidia flasks were inoculated and grown for 7-10 days, and subsequently used to inoculate an overnight 500mL culture grown 16 hours in Vogels medium and 1.5% sucrose. Cultures were harvested by Buchner funnel filtration, washed with dH2O, weighed, and frozen in liquid Nitrogen (LN2).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were harvested and added to a tube containing ~300uL 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 10 minutes at room temperature. Samples were cleared by centrifugation, and the aqueous (top) phase was added to 650uL cold 100% EtOH. Samples were precipitated at -20oC, pelleted by centrifugation, washed with 70% EtOH, and resuspended in DEPC-treated dH2O. RNA was treated with DNAse (DNA-free™ DNA Removal Kit, Thermo Fisher Scientific), cleaned (Agencourt® RNAclean XP® beads, Beckman Coulter), and RNA-seq libraries were prepared (KAPA Stranded mRNA-seq kit, KAPA Biosystems)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
polyA+ RNAseq libraires
|
| Data processing |
To begin, if data for the combined experiment (for both replicates) was to be analyzed, the replicate datasets were concatenated together with the "cat" command to make one .fastq file for each strain for each read direction (thus, two fastq files for each strain, since each paired-end experiment had R1 and R2 files; the combined files have the word "all" in the name). If the single replicates were to be analyzed independently, this step is omitted
To map the MNase-seq reads to the fixed version of the nc12 genome (Galazka et al)., paired end reads were mapped by bowtie with the following flags / parameters, per the protocols within the Nucwave program: "bowtie --suppress 1,6,7,8 -t --fr -p 6 -m 1 -v 2 -I 120 -X 180"; the -I and -X flags denote the distance between the two paired ends reads allowed for a mapped read pair to be recorded; the 120 to 180 basepair distance here is used to analyze only the mononucleosomes. The output file format is a .bowtie file.
The mapped paired-end read .bowtie file was run with the python program "nucwave_pe.py" with the "-w" flag to produce a .wig file. (instructions found at: http://nucleosome.usal.es/nucwave/, and are based on the manuscript: "Luis Quintales, Enrique Vázquez and Francisco Antequera (2015) Comparative analysis of methods for genome-wide nucleosome cartography. Briefings in Bioinformatics 16: 576-587.")
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 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.
For Bisulfite-seq files: The BRAT-BW software package (compbio.cs.ucr.edu/brat/; Harris et al, 2012) was used to prepare and map the reads to the N. crassa OR74A (annotation NC12) genome, which was converted to a four stranded reference genome to permit bisulfite mapping. BRAT-BW acgt-count “-B” option cytosine-only files produced for the forward and reverse strand reads were merged. The average 5mC level was determined for 25bp and 1bp step-wise window size across the genome using the Methpipe program (http://smithlabresearch.org/software/methpipe/). The resulting file was renamed with a “.igv” file extension to allow display on the Integrated Genome Viewer (IGV; www.broadinstitute.org/igv/)
For Whole-genome-sequencing files: Single nucleotide polymorphisms (SNPs) in parental and dim-1 strains, relative to the Neurospora version 10 genome, were identified using Freebayes (Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv preprint arXiv:1207.3907 [q-bio.GN] 2012), SNPs common between output vcf files were removed using vcf-isec within VCFtools (The Variant Call Format and VCFtools, Petr Danecek, Adam Auton, Goncalo Abecasis, Cornelis A. Albers, Eric Banks, Mark A. DePristo, Robert Handsaker, Gerton Lunter, Gabor Marth, Stephen T. Sherry, Gilean McVean, Richard Durbin and 1000 Genomes Project Analysis Group, Bioinformatics, 2011 ), the genic mutations of SNPs were identified using SnpEFF ("A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.", Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM. Fly (Austin). 2012 Apr-Jun;6(2):80-92.), and those residing on LG III were filtered using SnpSift ("Using Drosophila melanogaster as a model for genotoxic chemical mutational studies with a new program, SnpSift", Cingolani, P., et. al., Frontiers in Genetics, 3, 2012.).
For whole genome sequencing to build a reference genome: fasta file was built by mapping reads to the Neurospora crassa genome, version 10, whereupon the resulting sam file was converted to a bam file and sorted, and the genome sequence determined by samtools mpileup tool with the "-uf" flag, and the output was viewed with the "-cg -" flags, and converted from a vcf file to a fastq, and edited in TextEdit to a fasta file.
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 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.
Supplementary_files_format_and_content: For MNase-seq files: The .wig files are single nucleotide resolution of the nucleosome signal enrichment (read in number of reads) genome-wide that have been smoothed with the Nucwave program. Position .txt files for the nc12-fixed genome give the genome position, relative to each chromosome's left telomere, for either transcription start site (TSS) or the first nucleosome peak within that feature; position files are required as reference points to examine nucleosome position and enrichment changes.
Supplementary_files_format_and_content: For Bisulfite-seq files: .igv files displaying the % methylation over 25bp and 1bp windows where a score of 0 is a DNA region that has no cytosine methylation and a score of 1.0 is DNA where all of the cytosines are methylated in the region. Values in between 0 and 1.0 represent partial methylation of a region.
Supplementary_files_format_and_content: For whole genome sequencing, the parental strain: the N200 strain, which is the parental strain, has a .fasta file that provides its genome sequence
Supplementary_files_format_and_content: For whole genome sequencing: the mutant strains have vcf files that detail the SNPs found in LGIII, what gene they are located in, and the subsequent change to the coding sequence. These vcf files have intergenic and low-quality SNPs removed
Supplementary_files_format_and_content: For disiRNA-seq files: coordinates provided in GSE21175 were for the seventh version of the Neurospora genome. Data was downloaded and remapped to the nc12-fixed genome to make the .tdf file; the genome position coordinates for the Nc7 genome were reassigned to the locations in the nc12 version of the genome in the .bed file.
|
| |
|
| Submission date |
May 15, 2017 |
| 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 |
GPL20660 |
| Series (1) |
| GSE98911 |
Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora. |
|
| Relations |
| BioSample |
SAMN07118352 |
| SRA |
SRX2822430 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2627475_GATCAGAT_N5801_dim1_2_polyA_RNA_counts.txt.gz |
39.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
