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Sample GSM5135477 Query DataSets for GSM5135477
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) hidekit@uoregon.edu
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

Supplementary file Size Download File type/resource
GSM5135477_DamID_crf4-2_dam_set7_N8113.bw 2.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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