|
| Status |
Public on Sep 16, 2017 |
| Title |
6166_LexAO_HP1_K9me3_21617 |
| Sample type |
SRA |
| |
|
| Source name |
Mycelia
|
| Organism |
Neurospora crassa |
| Characteristics |
strain: N6166
chip antibody: anti-H3K9me3 (Active Motif, catalog# 39161, lot# 13509002)
genotype: his-3+::LexAO; HP1-LexADBD::hph
|
| Treatment protocol |
Mycelia were washed in PBS prior to crosslinking
|
| Growth protocol |
Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mycelia were cross-linked for 10 min in 0.5% formaldehyde for histone modification ChIP. Tissue was disrupted by sonication for thirty pulses (2x 15 pulses) before chromatin was sheared using a Bioruptor (Diagenode) for 15 min with a cycle of 30 sec on followed by 30 sec off, at high power. DNA was purified using the Qiagen Minelute reaction cleanup kit (Qiagen, 28606) and eluted in 50 μl of water.
Approximately 10 ng of DNA was used to generate ChIP-Seq libraries. Each library was prepared using the NEBNext Ultra DNA library prep kit (NEB #E7370) according to the manufacturer’s instructions. “Invisible” fragments between 225-425 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606). Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 9 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Tethered HP1 with LexAO binding site integrated at his-3.
|
| Data processing |
All processing steps were done utilizing the Galaxy bioinformatics suite (usegalaxy.org; Afgan et al. 2016). The raw data (fastq) files were converted to fastqsanger format using FASTQ groomer.
The converted fastq files were mapped to the Ncrassa12 genome using Bowtie2, generating .bam files
The bam files were then converted into tdf files using IGVtools. The Ncrassa12 genome was used as a reference genome and window size was set to 1 bp.
Genome_build: Ncrassa version 12 (fixed for inversion on LG VI) with LexAO sequence integrated (nc12_lexao.txt)
Supplementary_files_format_and_content: 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 1bp. The 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.
|
| |
|
| Submission date |
Sep 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Jordan David Gessaman |
| Organization name |
University of Oregon
|
| Department |
Biology, Institute of Molecular Biology
|
| Lab |
Selker
|
| Street address |
355 Streisinger Hall
|
| City |
Eugene |
| State/province |
OR |
| ZIP/Postal code |
97403 |
| Country |
USA |
| |
|
| Platform ID |
GPL23150 |
| Series (1) |
| GSE103926 |
Genome-wide maps of H3K9me3 with tethered heterochromatin machinery |
|
| Relations |
| BioSample |
SAMN07656231 |
| SRA |
SRX3190290 |
