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Sample GSM8152548

Query DataSets for GSM8152548

Status

Public on Mar 22, 2024

Title

BN1047, Rep1, Dam::BAF-1

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 400 ng genomic DNA extracted from 4000 L4 animals cultured and collected as indicated above. Three replicates were performed as described (de la Cruz Ruiz et al. 2022), using primer AdR (5'-NNNNGTGGTCGCGGCCGAGGATC) and 22 PCR cycles. Successful amplification of Dam-methylated genomic DNA yielded a smear of ~400-~1500 bp fragments when analyzed on Agilent Bioanalyzer. 400 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 pipelines RDamIDSeq (https://github.com/damidseq/RDamIDSeq) and RGeneDamIDSeq (https://github.com/damidseq/-RGeneDamIDSeq-) (Sharma et al. 2016) for mapping of reads to bins of fixed size (e.g. 2 kb, 10 kb or 100 kb) and to genes, respectively. The pipelines include 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 0.5 to 9 million (see Supplementary Table S7). Both pipelines also provide 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.
The DESeq2 package (Love et al. 2014) was used to identify bins with significantly more reads with either Dam::BAF-1 or Dam::BAF-1(G12T) compared to GFP::Dam across the 3 replicas. A false discovery rate (FDR) of 0.05 was used as threshold. Next, for each sample a pseudocount of 1 was added to each bin and the relative number of reads per bin was calculated followed by averaging across the replicas. The ratio of Dam::BAF-1 or Dam::BAF-1(G12T) to GFP::Dam was calculated for each bin and log2-transformed. Finally, normalization was done by subtracting the genome-wide average log2 ratio from the log2 ratio of each bin (Brueckner et al. 2020).
Assembly: ce11
Supplementary files format and content: Files contain columns for gene names (except in 2 kb bin files), coordinates, log2 values (Dam::BAF-1/GFP::Dam or Dam::BAF-1(G12T)/GFP::Dam)(average and individual replicas), bin (or gene) number and padj value.

Submission date

Mar 18, 2024

Last update date

Mar 22, 2024

Contact name

Peter Askjaer

E-mail(s)

pask@upo.es

Phone

+34 954 348 396

Organization name

Centro Andaluz de Biología del Desarrollo