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| Status |
Public on Sep 23, 2022 |
| Title |
Ribo-seq wild-type DD12 rep2 |
| Sample type |
SRA |
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| Source name |
mycelia
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| Organism |
Neurospora crassa |
| Characteristics |
genotype: wild-type
time point: DD12
replicate: 2
tissue: mycelia
molecule subtype: ribosome-protected mRNA
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| Growth protocol |
Mycelial disks in Vogel’s minimal media containing 2% glucose (pH 6.0) were synchronized to the same time of day by a shift from 30°C constant light (LL) to 25°C constant dark (DD). The cultures were grown in LL for a minimum of 4 h and transferred to DD on day 1 (for collection at DD 36, 40, 44, 48, 52), day 2 (for collection at DD 12, 16, 20, 24, 28, 32), day 3 (for collection at DD8), and harvested either at 9:00 a.m. (DD 12, 16, 20, 36, 40, 44) or 5:00 p.m. (DD 8, 24, 28, 32, 48, 52) on day 3.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Tissue was harvested in the dark, pressed in between paper towels to remove excess media, and immediately frozen in liquid N2. Ribosomes were pelleted by sucrose-density centrifugation and ribosome-protected mRNAs were isolated from the pelleted cell extracts using the Qiagen miRNeasy Mini Kit (Cat. # 217004).
All subsequent steps up to step 46 in the published ribosome profiling protocol (Ingolia et al., 2012) were followed as described. An rRNA depletion step was not performed, but instead after heat-inactivation of CircLigase, 2.0 μL of GlycoBlueTM, 6.0 μL 5 M NaCl, 74 μL of DEPC water, and 150 μL of isopropanol were added to each tube and precipitation was carried out overnight at -80°C. The DNA was pelleted by centrifugation for 30 min at 20,000 g at 4°C. The pellets were washed with 70% ethanol and allowed to air-dry for 10 min. The pellet was then resuspended in 5.0 μL of 10 mM Tris (pH 8.0), and cDNA synthesis using SuperScript™ III Reverse Transcriptase, followed by barcode addition by PCR amplification using Phusion Hot Start High-Fidelity DNA Polymerase were performed as described (101). Sequencing libraries were quantified and checked for quality on a 2100 Bioanalyzer using a DNA high sensitivity chip per the manufacturer’s instructions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
rp_wt2_12_HTS2617
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| Data processing |
Library strategy: Ribo-seq
For WT, Dfrq, and Dcpc-3 samples, basecalling was performed using the Real Time Analysis (RTA) workflow ver. 2.7.7. with default parameters. For cpc-3c samples, basecalling was performed using Illumina BaseSpace app using the FASTQ generation application ver. 1.0.0 and bcl2fastq.
Ribosome profiling (ribo-seq) reads were trimmed using Cutadapt (Martin, 2011) to remove the 3′ adapter of the reads (adapter sequence – CTGTAGGCACCATCAAT).
Adapter-trimmed ribo-seq reads were size-filtered to the 28-34 nt range using Trimmomatic (Bolger et al., 2014).
Ribo-seq reads were aligned to the N. crassa genome FungiDB Release 38 using STAR (Dobin et al., 2013).
To calculate the ribo-seq read coverage over the 5’ UTR, 3’ UTR, or CDS regions, the geneBody_coverage2.py program from the RSeQC package (Wnag et al., 2012) was used.
Ribosome-protected footprint (RPF) features such as read length distribution and sub-codon phasing were determined using NGS toolkit Plastid (Dunn et al, 2016).
The RPF originating only from the coding sequences (CDS) were extracted and quantified using HTSeq-count (Anders et al., 2015). The counted reads were normalized using the median of ratios method employed in the DESeq2 analysis workflow (Love et al., 2014).
The normalized RPF time series was analyzed using the following Extended Circadian Harmonic Oscillation (ECHO, De Los Santos et al., 2019) settings: two unpaired replicates, smoothing function enabled, unexpressed genes excluded from the analysis at the default 70% threshold. Genes were considered rhythmic if they had Benjamini-Hochberg p-values < 0.05, and oscillation types harmonic, damped or forced.
Genome_build: N. crassa genome FungiDB Release 38
Supplementary_files_format_and_content: Output from normalized HTSeq-count done on the reads originating from the CDS and saved as plain text files containing ribosome-occupancy reads.
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| Submission date |
Aug 06, 2021 |
| Last update date |
Sep 25, 2022 |
| Contact name |
Deborah Bell-Pedersen |
| E-mail(s) |
dpedersen@bio.tamu.edu
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| Organization name |
Texas A&M University
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| Department |
Biology
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| Street address |
3258 TAMU
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| City |
College Station |
| State/province |
Texas |
| ZIP/Postal code |
77840 |
| Country |
USA |
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| Platform ID |
GPL30082 |
| Series (2) |
| GSE181565 |
Circadian Clock-Controlled Translation of Specific mRNAs in Neurospora crassa Requires Rhythmic eIF2α Activity and P-body Sequestration [Ribo-seq] |
| GSE181566 |
Circadian Clock-Controlled Translation of Specific mRNAs in Neurospora crassa Requires Rhythmic eIF2α Activity and P-body Sequestration |
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| Relations |
| BioSample |
SAMN20601296 |
| SRA |
SRX11661826 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5506275_rp_wt2_12_HTS2617_norm_cdsreads.txt.gz |
61.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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