|
| Status |
Public on Mar 21, 2024 |
| Title |
BN561, Rep1, GFP::Dam, RAPID |
| Sample type |
SRA |
| |
|
| Source name |
Hypodermis
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: Hypodermis
genotype: WT
|
| Growth protocol |
Worms were grown asynchronically at 20C on NGM plates seeded with E. coli GM119 for at least two generations before egg-prepping with hypochlorite. Hatched L1s were counted the day after and 1000 L1s were aliquoted onto 10 cm NGM plates seeded with E. coli GM119 as described above. Worms were grown at 20C for 48 h before collecting 4000 L4 stage nematodes per strain and washed 8 times with 15 ml of M9 with 0.01% Tween-20. Worms were transferred to 1.5 ml tubes with M9 with 0.01% Tween-20 using Pasteur pipettes followed by two additional washes. Supernatants were aspirated and aliquots of 30 µl were snap frozen in liquid nitrogen and maintained at -80C until DNA extraction.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
To purify genomic DNA, samples were lysed in 5 freeze/thaw cycles (1min in liquid nitrogen; 3min in 37C shaker) and processed with DNeasy Blood and Tissue Kit (QIAGEN #69504).
DamID was performed on 200 ng genomic DNA extracted from 4000 L4 animals cultured and collected as indicated above. Two replicates were performed as described (de la Cruz Ruiz et al. 2022). A pool of 3 AdR primers (5'-N(4-6)GTCCTCGCGGCCGAGGATC) and 14 PCR cycles were used. Successful amplification of Dam-methylated genomic DNA yielded a smear of ~400-~1500 bp fragments when analyzed on Agilent Bioanalyzer. 40 ng of PCR amplified fragments were used for library preparation as described (de la Cruz Ruiz et al. 2022), using E7370 NEBNext® Ultra™ DNA Library Prep Kit for Illumina. Library amplicons’ size distribution was checked to be maintained as for PCR fragments indicated above followed by pooling, repurification and sequencing on a Illumina NextSeq500 platform at EMBL GeneCore.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
FASTQ files from the NextSeq500 platform were processed in R Studio using the pipeline RGeneDamIDSeq (https://github.com/damidseq/-RGeneDamIDSeq-) (Sharma et al. 2016) for mapping of reads to genes. The pipeline includes a quality check where only reads that contain the DamID adapter (“CGCGGCCGAG”) and map to GATC sites in the C. elegans genome are retained for further analysis. The number of reads fulfilling these criteria ranged from 7.2 to 27 million. The pipeline also provides mapped read numbers per individual GATC fragment. For mapping to genes, the script was modified to include 500 bp distal to transcriptional start and termination sites. UCSC genome version ce11 (BSgenome.Celegans.UCSC.ce11) was used for alignment.
Normalization of Dam::RPB-6 reads to GFP::Dam reads was performed using the damidseq_pipeline package (https://owenjm.github.io/damidseq_pipeline/) (Marshall and Brand 2015) on the bam files generated by the RDamIDSeq pipeline. Gene occupancy was computed as log2(Dam::RPB-6/GFP::Dam) with polii.gene.call (https://github.com/owenjm/polii.gene.call) using bedgraph output files from the damidseq_pipeline and WBCel235 gene annotation. A FDR of 0.05 was used as threshold to call transcribed genes.
Assembly: ce11
Supplementary files format and content: csv file polii.gene.call from script; contains for each gene log2 score, number of GATC fragments and FDR.
|
| |
|
| Submission date |
Mar 18, 2024 |
| Last update date |
Mar 21, 2024 |
| Contact name |
Peter Askjaer |
| E-mail(s) |
pask@upo.es
|
| Phone |
+34 954 348 396
|
| Organization name |
Centro Andaluz de Biología del Desarrollo
|
| Street address |
UPO Carretera de Utrera km 1
|
| City |
Seville |
| ZIP/Postal code |
41013 |
| Country |
Spain |
| |
|
| Platform ID |
GPL19757 |
| Series (1) |
| GSE261819 |
A progeria-associated BAF-1 mutation modulates gene expression and accelerates aging in C. elegans I |
|
| Relations |
| BioSample |
SAMN40530174 |
| SRA |
SRX23981582 |
