|
| Status |
Public on Mar 08, 2022 |
| Title |
DamID_crf4-2_dam_set7_N8113 |
| Sample type |
SRA |
| |
|
| Source name |
nuclei from germinated condia
|
| Organism |
Neurospora crassa |
| Characteristics |
sample type: nuclei
|
| Treatment protocol |
Mycelia were harvested, lyophilized, pulverized and resuspended in salt detergent. Lysates were cleared by centrifugation and TCA/ethanol treatment. Total genomic DNA was isolated by phenol-chloroform extraction and isopropanol precipitation and resuspended in TE buffer
|
| Growth protocol |
5 ml cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 24 hours at 32oC.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Preparation of N6-methyladenine-containing DNA for sequencing was performed using a previously reported procedure (Bernstein et al. 2012) 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) and prepared for high-throughput sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
DamID
|
| Data processing |
DamID-seq mapping and analysis was done using the Galaxy public server (Afgan et al., 2018).
The Barcode Splitter was used to filter for reads with a GATC at the 5’ end and these reads were mapped using Bowtie2 (Langmead and Salzberg, 2012).
Files for biological replicates were merged using MergeBam.
Merged bam files were used as input for bamCoverage (RPKM, 50bp bins) to generate bigwig files for viewing on IGV and running bigwigCompare.
The output from bamCoverage was used with computeMatirx to generate files to use for plotProfile and output graphs.
neurospora crassa genome (OR74A)
bigwig
|
| |
|
| Submission date |
Mar 04, 2021 |
| Last update date |
Mar 08, 2022 |
| Contact name |
Hideki Tanizawa |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Oregon
|
| Department |
Institute of Molecular Biology
|
| Street address |
1370 Franklin Blvd
|
| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
| |
|
| Platform ID |
GPL23150 |
| Series (1) |
| GSE168277 |
The ACF chromatin remodeling complex is essential for Polycomb repression |
|
| Relations |
| BioSample |
SAMN18141470 |
| SRA |
SRX10240512 |
