|
| Status |
Public on Oct 05, 2021 |
| Title |
NLS-GFP:dam_L4_rep-2_Exp-1 |
| Sample type |
SRA |
| |
|
| Source name |
Epidermis
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: MBA444
tissue: Epidermis - wrt-2 expression domain
developmental stage: L4
biological replicate: Replicate 2
experiment number: 1
sequenced molecule: DNA amplicons
|
| Growth protocol |
For each replicate of each strain used for TaDa nine 55 mm NGM plates seeded with dam-/dcm- bacterial lawns and fully populated by gravid adults were used for large-scale egg preparation with isolated embryos seeded in new dam-/dcm- plates that were incubated at 20 °C. Half of the resulting synchronised populations were collected in a 15 ml centrifuge tube after 24 hours at the L2 stage and the other half after 48 hours at the L4 stage. All collected populations pellets (~100 μl) underwent extensive washing with 10 ml M9 at least 5 times.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
gDNA for each sample was extracted by lysing animal pellets with Cell Lysis Solution (QIAGEN) containing Proteinase K. RNA was removed by treating with RNase A and DNA was isolated after treatment with Protein precipitation solution (QIAGEN) by percipitating in isopropanol and washing with 70% ethanol. gDNA was redissolved in water. For isolation and amplification of the GATC methylated gDNA fragments the protocol followed was an adapted version of the one presented in (Marshall et al., 2016)
Amplicon sonication and library preparation was outsourced to GENEWIZ which used illumina protocols for sequencing on the Illumina HiSeq 2x150 platform
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| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
amplicons of GATC-6mA-methylated genomic DNA fragments
|
| Data processing |
Library strategy: DamID-seq
Sequencing data were fully processed using the automated pipeline defined by the perl script damidseq_pipeline v1.4.5 (Marshall and Brand, 2015) (available at https://github.com/owenjm/damidseq_pipeline). The pipeline was used calling Bowtie 2 v2.3.4 (Langmead and Salzberg, 2012) for alignment to C. elegans bowtie indices from genome assembly WBcel235 (available from illumina® iGenomes page), Samtools v1.9 (Li et al., 2009) for alignment manipulations and generation of BAM files. Using a GATC fragment file with the coordinates of all GATC fragments across the C. elegans genome in gff format, built from a WBcel235 FASTA file (available at https://www.ensembl.org/Caenorhabditis_elegans/Info/Index) using the gatc.track.marker.pl script (available at https://github.com/owenjm/damidseq_pipeline), the pipeline calculated genome-wide log2(TF:dam/NLS-GFP:dam) signal enrichment scores per GATC fragment of the genome as gff files from pairs of BAM files from TF:dam and NLS-GFP:dam samples.
Using damidseq_pipeline v1.4.5 all pairwise calculations of log2(TF:dam/NLS-GFP:dam) scores between pairs of TF:dam and NLS-GFP:dam samples replicates from the same experiment and developmental stage were performed producing 4 independet gff signal files for each TF at each stage.
The 4 gff signal files were averaged arithmetically per GATC fragment of the genome to genarate the single averaged signla gff file each TF at each developmental stage.
Statistically significant enrichment peaks with an FDR<0.05 were identified from the averaged profiles using the the perl script find_peaks (available at https://github.com/owenjm/find_peaks) which generated peak interval gff files
Genome_build: WBcel235
Supplementary_files_format_and_content: GFF interval files representing coordinates of GATC fragments of the genome with a log2(TF:dam/NLS-GFP:dam) score each
*averaged.gatc.chr.gff = averaged log2 signal across replicates per GATC fragment
*peaks.gff = scored peak files
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| |
|
| Submission date |
Dec 15, 2020 |
| Last update date |
Oct 05, 2021 |
| Contact name |
Dimitris Katsanos |
| Organization name |
Imperial College London
|
| Department |
Life Sciences
|
| Lab |
Michalis Barkoulas
|
| Street address |
Sir Alexander Fleming building, Imperial College Road
|
| City |
London |
| ZIP/Postal code |
SW7 2AZ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL22765 |
| Series (1) |
| GSE163243 |
Targeted DamID in C. elegans reveals a direct role for LIN-22 and NHR-25 in antagonising the epidermal stem cell fate |
|
| Relations |
| BioSample |
SAMN17084540 |
| SRA |
SRX9686702 |
