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| Status |
Public on Jan 09, 2023 |
| Title |
HPL-2-Dam_hypodermis_rep1 |
| Sample type |
SRA |
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| Source name |
hypodermis
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| Organism |
Caenorhabditis elegans |
| Characteristics |
tissue: hypodermis
developmental stage: L4 larvae
genotype: MRS578 (HPL-2::Dam in hypodermis)
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| Growth protocol |
For synchronization of nematodes by hypochlorite treatment (“bleaching”), a mixed-stage population with many gravid adults was washed off an NGM plate with M9 into a 15 mL tube and centrifuged at 500g during 2 min. The supernatant was aspirated and the animals resuspended in 15 mL M9 buffer and centrifuged again at 500 g for 2 min. The washing was repeated and the supernatant aspirated as much as possible without disturbing the pellet of worms. Two mL of hypochlorite solution was added and shaken. The animals were monitored under a stereoscope until the majority were broken up (this depends on the concentration of the hypochlorite; should not be more than 5–6 min) and the tubes were filled immediately with sterile M9 and centrifuged at 800 g for 1 min. The supernatant was aspirated and the embryos resuspended in 15 mL M9 buffer and centrifuged again at 800 g for 1 min. The washes were repeated ten times (in sterile conditions) and left in a suitable volume of M9 to reach ~20 embryos/μL. Animals were incubated for 48h at 20°C until L4 stage. Larvae were harvested by centrifugation, washed with M9 10 times and distributed in aliquots of 30 μL. Excess liquid was removed and aliquots were snap-frozen in liquid nitrogen and stored at -80°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA (gDNA) extraction from ~30 μl L4s was performed by 5 rounds of rapid freeze/thaw cycles in liquid nitrogen and 37°C water bath to break the worm cuticle followed by purification using a DNeasy Blood and Tissue Kit (QIAGEN #69504). gDNA was eluted in two rounds in a total volume of 300 μl and precipitated using 0.1x volume NaAc 3M and 2.5x volume 96% ethanol. The final concentration after resuspension in 10 μl of 10mM Tris-Cl was measured by QUBIT.
To prepare the libraries, 200 ng gDNA were digested using 10 units DpnI (NEB #R0176S; cuts methylated GATC) in 10 μl for 6 hours at 37°C in a thermocycler. Next, DpnI was inactivated by heating to 80°C for 20 min. Double-stranded adaptors were prepared by heating a mixture of 50 μl primer AdRt (5’-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA, 100 μM) and 50 μl AdRb (5’-TCCTCGGCCGCG; 100 μM) to 95°C and cooling slowly to room temperature. Next, 0.8 μl of the double-stranded adapters were ligated to the DpnI-digested gDNA in a volume of 20 μl at 16°C in a thermocycler overnight using T4 DNA ligase (Roche #10799009001), which later was inactivated by incubation for 10 min at 65°C. The ligated gDNA was purified with 1.8x volume Agencourt AMPure XP® beads (Beckman Coulter; #A63880) using a magnetic particle concentrator, washed with 180 μl of freshly prepared 70% ethanol and eluted in 20 μl elution buffer (EB; 10 mM Tris-Cl, pH 8.5). Unmethylated GATC sites were digested with 10 units of DpnII (NEB #R0543S) in a volume of 50 μl for 1 h at 37°C. Next, DpnII was inactivated by heating to 80°C for 20 min. The digested gDNA was re-purified with AMPure XP® beads as above and eluted in 25 μl. Methylated gDNA fragments were amplified using Taq DNA polymerase with ThermoPol buffer (NEB #M0267) in 50 μl reactions containing 5 μl 10x PCR reaction buffer, 25 μl DpnII digested DNA, 1.25 μl primer AdR (5’-NNNNGTGGTCGCGGCCGAGGATC; 50 μM), 4 μl dNTP mix (each 2.5 mM), 1 μl polymerase. PCR parameters were 1) 68°C 10 min; 2) 94°C 1 min; 3) 65°C 5 min; 4) 68°C 15 min; 5) 94°C 1 min; 6) 65°C 1 min; 7) 68°C 10 min; 8) Go to step 5 and repeat three times; 9) 94°C 1 min; 10) 65°C 1 min; 11) 68°C 2 min; 12) Go to step 9 and repeat 19 times. Five μl of each PCR reaction were analyzed by agarose gel electrophoresis: successful amplification of methylated DNA was confirmed by the presence of a 400–1000 bp smear which was not observed in controls without DpnI or T4 DNA ligase. Finally, the PCR products were purified using QIAquick PCR Purification Kit (QIAGEN #28104), eluted in 30 μl EB and their concentration was measured using QUBIT. 400 ng of PCR fragments were used as input in the NEBNext Ultra DNA Library Prep Kit for Illumina (E7370). The manufacturer’s instructions were used with the following conditions. Size selection was performed by first adding 30 μl of resuspended SpeedBead Magnetic Carboxylate Modified Particles, Azide 0.05% (GE65152105050250) to the 100 μl ligation reaction. The beads were discarded and the supernatant was mixed with 60 μl resuspended SpeedBeads. Then, the supernatant was discarded and the beads were washed with ethanol and DNA was eluted in 17 μl of 10mM Tris. Eight PCR cycles were performed to amplify the samples. The quality of the samples was verified by agarose gel electrophoresis and QUBIT analysis as above.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The quality control of the DamID technique was performed by damid.seq.r pipeline version 0.1.3 in Rstudio (R version 3.4.3) (available at github.com/damidseq/rDamlDSeq) (Sharma, Ritler, and Meister 2016). From FastQ files, the pipeline detects and maps reads containing the DamID adapters (adapt.seq="CGCGGCCGAG”) and match them to genomic regions flanked by GATCs sites (restr.seq = "GATC") to obtain Bam Files. The Bam files were used to generate the normalized log2 HPL::Dam/GFP::Dam ratio scores per GATC fragments, using perl script damidseq_pipeline v1.4.5 (Marshall and Brand 2015) (available at https://github.com/owenjm/damidseq_pipeline). The pipeline integrates a Bowtie 2 v2.3.4 (Langmead and Salzberg 2012) calling to map reads on C. elegans bowtie indexes from assembly WBcel235 (available in Illumina iGenomes webpage; identical to ce11). In addition, it includes also Samtools v1.9 (Li et al. 2009) for alignment guidance, and a GFF file containing all GATC sites in the C. elegans genome. Last GATC coordinates file was built by gatc.track.maker.pl tool provided by the same pipeline script using WBcel235 FASTA file for C. elegans (available at https:// ensembl.org/Caenorhabditis_elegans/Info/Index. Final bedgraph files for each replica was averaged using average_tracks perl script included in damid_misc (available at https://github.com/owenjm/damid_misc).
The selection of statistically significant peaks for every HP1 sample was done by find_peaks perl script (available at https://github.com/owenjm/find_ peaks) with a false discovery rate (FDR) lower than 0.05 using as input averaged bedgraph files containing normalized log2 HPL::Dam/GFP::Dam ratio scores per GATC fragment.
Assembly: ce11
Supplementary files format and content: bedgraph files from damidseq_pipeline script; 1 file per replica and 1 file with combined data; contain log2 scores for each GATC fragment.
Supplementary files format and content: gff file from find_peaks script, 1 file per combined data that contains all peaks with an FDR less than 0.05.
Library strategy: DamID-seq
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| Submission date |
Jan 03, 2023 |
| Last update date |
Jan 10, 2023 |
| Contact name |
Patricia De La Cruz Ruiz |
| E-mail(s) |
[email protected]
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| Organization name |
1Andalusian Centre for Developmental Biology. Universidad Pablo de Olavide
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| Street address |
Ctra. de Utrera, km 1
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| City |
Seville |
| ZIP/Postal code |
41013 |
| Country |
Spain |
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| Platform ID |
GPL19757 |
| Series (1) |
| GSE222056 |
Tissue-specific chromatin binding patterns of C. elegans heterochromatin proteins HPL-1 and HPL-2 reveal differential roles in the regulation of gene expression. |
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| Relations |
| BioSample |
SAMN32541601 |
| SRA |
SRX18912214 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6913973_MRS578R3-cut_4b21f5f12d6-vs-Dam.gatc.average-FDR0.05.peaks.gff.gz |
20.5 Kb |
(ftp)(http) |
GFF |
| GSM6913973_MRS578R3-cut_4b21f5f12d6-vs-Dam.gatc.average.bedgraph.gz |
3.5 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM6913973_MRS578R3-cut_4b21f5f12d6-vs-Dam.gatc.bedgraph.gz |
3.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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