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| Status |
Public on Nov 08, 2021 |
| Title |
lancelet_larvae_ACEseq_rep2 |
| Sample type |
SRA |
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| Source name |
lancelet larvae DNA subjected to ACE-seq rep2
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| Organism |
Branchiostoma lanceolatum |
| Characteristics |
age: larvae
tissue: whole organism
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ACE-seq library preparation for the DNA hydroxymethylation profiling was performed as previously described {Schutsky et al, 2018 PMID: 30295673;Wang et al, 2021 PMID: 32822044}. Briefly, 100 ng of genomic DNA extracted from sea urchin, lancelet and zebrafish embryos and adult tissues was spiked with 1ng of CpG methylated λ phage DNA (Wisegene) and 0.5 ng of all-C hydroxymethylated pUC19 plasmid DNA (Wisegene) and sonicated to ~300 bp fragments using a M220 focused ultrasonicator (Covaris) with the following parameters: peak incident power, 50W; duty factor, 20%; cycles per burst, 200; treatment time, 75 sec. Sonicated DNA (125 ul total volume) was concentrated to 16.6 ul using AMPure beads (1 volume DNA : 2 volumes AMPure beads). 5hmC protection reaction was performed using β-glucosetransferase (10U/ul, New England Biolabs) in Cutsmart Buffer according to manufacturer’s instructions at 37°C for 1 hr, followed by 10 min at 65°C. The DNA was denatured in DMSO at 95°C for 10 min and immediately placed on dry ice for 2 mins allowing the samples to freeze. C and 5mC deamination reaction was performed using the APOBEC3A enzyme (NEBNext® Enzymatic Methyl-seq Kit, New England Biolabs) with the following ramping conditions: 4°C for 10 min, 4°C - 50°C 2:15 min per degree of the ramp, 50°C for 10 min. Deaminated DNA was then purified using AMPure beads (1 volume DNA : 1 volume AMPure beads) and subjected to low input library preparation using Accel-NGS Methyl-Seq DNA kit (Swift Biosciences). Briefly, DNA was denatured and subjected to an adaptase reaction, followed by primer extension, adapter ligation, and 15 cycles of indexing PCR. Library size and consistency was determined by the Agilent 4200 Tapestation system. The libraries were quantified using the KAPA library quantification kit (Roche).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: ACE-seq
Adapter clipping and read quality trimming using Trimmomatic (ILLUMINACLIP:NEXTflex.fa:2:30:10 SLIDINGWINDOW:5:20 LEADING:5 TRAILING:5 MINLEN:50 CROP:140 HEADCROP:10) (Bolger et al, 2014)
Trimmed reads were mapped to Spur_v3.1, Bl71nemr, danRer10 and mm10 genome references for sea urchin, lancelet, zebrafish and mouse, respectively, (all containing the λ phage genome as chrLambda and pUC19 plasmid genome as chrPUC) using WALT (Chen et al, 2016) with the following settings: -m 10 -t 24 -N 10000000 -L 2000.
Optical and PCR duplicates were removed using Picard Tools’s function MarkDuplicates REMOVE_DUPLICATES=true (http://broadinstitute.github.io/picard/).
Reads with more than three consecutive non-converted cytosines in the CC, CA and CT context were removed using Picard Tools’ function FilterSamReads, in order to eliminate DNA molecules not efficiently deaminated by APOBEC3A
The numbers of hydroxymethylated and unmethylated cytosines at each genomic CpG position were called using the MethylDackel extract genome.fasta input.bam -o output --mergeContext. Additional MethylDackel extract parameters --minOppositeDepth 5 --maxVariantFrac 0.5 were used for the genotype correction (in order to exclude C-to-T nucleotide transitions in the genomic DNA of interest, which could be incorrectly considered as unmodified cytosines) (https://github.com/dpryan79/MethylDackel)
Genome_build: sea urchin: Spur_v3.1; lancelet: Bl71nemr; zabrafish: danRer10
Supplementary_files_format_and_content: bed / The file contains 1. Chromosome / Scaffold; 2. CpG start coordinate; 3. CpG end coordinate; 4. The methylation percentage rounded to an integer; 5. The number of alignments/pairs reporting hydroxymethylated bases; 6. The number of alignments/pairs reporting unmethylated bases
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| Submission date |
Nov 06, 2021 |
| Last update date |
Nov 08, 2021 |
| Contact name |
Ksenia Skvortsova |
| E-mail(s) |
k.skvortsova@garvan.org.au
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| Phone |
478135210
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| Organization name |
Garvan Institute
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| Department |
Genomics and Epigenetics
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| Lab |
Developmental Epigenomics
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| Street address |
384 Victoria St, Darlinghurst
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| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
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| Platform ID |
GPL27879 |
| Series (2) |
| GSE188331 |
Base-resolution DNA hydroxymethylation maps of purple sea urchin (Strongylocentrotus purpuratus), european lancelet (Branchiostoma lanceolatum) and zebrafish (Danio rerio) using ACE-seq |
| GSE188334 |
Base-resolution 5-hydroxymethylcytosine maps of sea urchin and lancelet embryos and adult tissues |
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| Relations |
| BioSample |
SAMN22958221 |
| SRA |
SRX13025572 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5677926_BLrep2_larvae_S8_L004_Bl71nemr_sorted_dedup_sorted_filter_CpG_cov10.bed.gz |
41.1 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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