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| Status |
Public on Jun 10, 2024 |
| Title |
NKL2_LGIIK9het25-deletion_polyA-mRNA-seq_second-rep |
| 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
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| Treatment protocol |
Cultures were harvested by filtration and washed with 1xPBS prior to the RNA isolation protocol
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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 |
polyA RNA |
| Extraction protocol |
Cultures were harvested by vacuum filtration, placed in 2.0mL screw-top tubes, and 300µL of acid washed glass beads suspended in dH2O (150-212µm, Sigma Aldrich cat# G1145-10G), 350µL Acid Phenol:chloroform (5:1; ThermoFisher cat# AM9720), and 350µL NETS (300mM NaCl, 1mM EDTA, 10mM Tris pH 7.5, 0.2% SDS) were added. Samples were vortexed for 10 minutes at maximum speed and centrifuged at 4oC for 5 minutes at 13k rpm. The aqueous layer was transferred to two tubes (~250µL each), 650µL of cold 100% EtOH was added, and RNA was precipitated on ice for 10 minutes and pelleted by microcentrifugation at 4oC for 10 minutes at 13k rpm. The pellet was washed with cold 70% EtOH (made with DECP-treated dH2O), the supernatant was removed, and pellets were allowed to air dry for 15 minutes and resuspended in 50µL DEPC-treated dH2O. Insoluble material was removed by centrifugation, and the initial RNA concentration was measured by Nanodrop. Total RNA (10µg) was resuspended in 50µL of DEPC-treated dH2O. Total RNA cleanup was performed by the Zymo RNA clean and concentrator kit (Zymo Research cat# R1013), per the manufacturer’s protocol; the 15µL elution was divided into 4µL for Qubit concentration (RNA HS assay kit; ThermoFisher Scientific, cat# Q32852) and TapeStation (Agilent) quality control analysis, while the remaining 11µL was stored at -80oC. Only total RNA samples with high rRNA:mRNA ratios were used for library construction.
Polyadenylated messenger RNA (polyA mRNA) was selected with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs [NEB], cat# E7490L) and polyA mRNA sequencing libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, cat# E7760L and E7765L) per the manufacturer’s protocols; quadruplicate polyA mRNA-seq replicate libraries were constructed for each strain. All polyA mRNA-seq libraries were Illumina sequenced at the GC3F on a NovaSeq 6000 as SR118 reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
matrix column names: column1 = Neurospora crassa gene name; column2 = gene count value
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| Data processing |
Raw fastq files were mapped to the nc14 genome using HiSat2 (72), and gene count files were generated with htseq-count (73). Differential Expression analysis between strains was performed with DESeq2 (74) in R/Rstudio (75, 76), selecting significantly changed genes as those with expression log2 > 3.0 or log2 < -3.0 and an adjusted p-value < 0.001; bed files for display on IGV were generated from the DESeq2 output gene lists in Microsoft Excel, and volcano plots of log2 fold change vs. adjusted p-value were generated in R/R studio.
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: htseq-count files are two column files with the first column being the gene name (specific to Neurospora crassa; NCU#), the second column being the count value of that gene
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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) |
| GSE269230 |
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa [RNA-Seq] |
| GSE269235 |
A Constitutive Heterochromatic Region Shapes Genome Organization and Impacts Gene Expression in Neurospora crassa |
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| Relations |
| BioSample |
SAMN41708019 |
| SRA |
SRX24823196 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8309035_NKL2_delta-LGIIK9het25_polyA-mRNA_rep3_hisat2_counts_not-stranded.txt.gz |
39.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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