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| Status |
Public on Apr 15, 2020 |
| Title |
EPR-1PHD N7901 DAM-ID-seq |
| Sample type |
SRA |
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| Source name |
germinated conidia
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| Organism |
Neurospora crassa |
| Characteristics |
genotype: mat a; epr-1W292A::10xGly::Dam::nat-1
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Preparation of N6-methyladenine-containing DNA for sequencing was performed using a previously reported procedure {Bernstein:2012wx} with the following modifications: 5 μg of genomic DNA from N. crassa strains expressing a Dam fusion was digested with 1 μL of DpnI (NEB, 20 units/μL); ligation to primer 5050 was carried out overnight at 16 °C; amplification reactions of ligated DNA with primer 5051 were performed in triplicate, contained 5 μL dNTPs, and an additional PCR cycle was added to the 2nd and 3rd phase of the PCR protocol (4 and 18 cycles respectively); 3 μg of pooled, amplified DNA was sheared using a Bioruptor (Diagenode) twice on high for 10 minutes (30 seconds on/off) at 4 °C; biotinylated DNA was purified using 250 μL slurry of streptavidin-agarose beads (Sigma).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
GCTCAGTT_N7901_WT_NCU07505_W292A-DAM_122018_S63_L008_R1_001
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| Data processing |
Library strategy: DamID-seq
ChIP-seq processing: All sequencing reads were mapped to the corrected N. crassa OR74A (NC12 genome) (Galazka et al., 2016) using Bowtie2 (Langmead and Salzberg, 2012). ChIP-seq read coverage was averaged, normalized, and analyzed using tools available from deepTools2 on the open-source platform Galaxy (Afgan et al., 2016). Sequencing tracks are displayed as 25-nt-window TDF or bigWig files with the Integrative Genomics Viewer (IGV) (Robinson et al., 2011).
RNA-seq processing: Tools available on Galaxy {Afgan:2018gf} were used to map mRNA-sequencing reads (intron size < 1kb) {Dobin:2012fg} against the corrected N. crassa OR74A (NC12) genome {Galazka:2016cm}, count the number of reads per gene {Dobin:2012fg} and determine differentially expressed genes with DESeq2 {Love:2014ka}.
DamID-seq processing: All sequencing reads were mapped to the corrected N. crassa OR74A (NC12 genome) (Galazka et al., 2016) using Bowtie2 (Langmead and Salzberg, 2012). ChIP-seq read coverage was averaged, normalized, and analyzed using tools available from deepTools2 on the open-source platform Galaxy (Afgan et al., 2016). Sequencing tracks are displayed as 25-nt-window TDF or bigWig files with the Integrative Genomics Viewer (IGV) (Robinson et al., 2011).
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: ChIP-seq processed data files are bigwig files generated using DeepTools (Ramirez et al., 2016) using a window size of 25bp
Supplementary_files_format_and_content: RNA-seq processed data files are txt files containing normalized counts for 2 replicates each of wild type, ∆set-7 (both from Klocko et al. 2016; GSE82222) and ∆epr-1 and the output file from DESeq2 (Love et al., 2014) with pairwise comparisons of 2 biological replicates from each genotype.
Supplementary_files_format_and_content: DamID-seq processed data files are bigwig files generated using DeepTools (Ramirez et al., 2016) using a window size of 25bp
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| Submission date |
Mar 14, 2019 |
| Last update date |
Apr 15, 2020 |
| Contact name |
Elizabeth Toomey Wiles |
| E-mail(s) |
[email protected]
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| Organization name |
University of Oregon
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| Department |
Biology, Institute of Molecular Biology
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| Lab |
Selker
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| Street address |
1229 University of Oregon; 1318 Franklin Blvd.
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| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
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| Platform ID |
GPL23150 |
| Series (1) |
| GSE128317 |
Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing in Neurospora crassa |
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| Relations |
| BioSample |
SAMN11128299 |
| SRA |
SRX5523373 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3671341_GCTCAGTT_N7901_WT_NCU07505_W292A-DAM_122018_S63_L008_R1_001.bigwig |
4.3 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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