|
| Status |
Public on Nov 08, 2021 |
| Title |
seaurchin_24hpf_embryo_MethylCseq_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
24 hpf sea urchin embryo DNA subjected to MethylC-seq rep1
|
| Organism |
Strongylocentrotus purpuratus |
| Characteristics |
tissue type: 24 hpf embryo
tissue: embryo
|
| Treatment protocol |
no treatment
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MethylC-seq library preparation was performed as described previously (PMID: 25692984). Briefly, 1000 ng of genomic DNA extracted from sea urchin and lancelet embryos and adult tissues was spiked with unmethylated λ phage DNA (Promega) and sonicated to ~300 bp fragments using 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 was then purified, end-repaired using End-It™ DNA End-Repair Kit (Lucigen) and A-tailed using Klenow Fragment (3'→5' exo-) (New England Biolabs) followed by the ligation of NEXTFLEX® Bisulfite-Seq Adapters. Bisulfite conversion of adaptor-ligated DNA was performed using EZ DNA Methylation-Gold Kit (Zymo Research). Library amplification (13 PCR cycles) was performed with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems). Library size was determined by the Agilent 4200 Tapestation system. The libraries were quantified using the KAPA library quantification kit (Roche) yielding ~10-20 nM.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Sequenced reads in fastq format were trimmed using Trimmomatic software (ILLUMINACLIP:adapter.fa:2:30:10 SLIDINGWINDOW:5:20 LEADING:3 TRAILING:3 MINLEN:50)
Trimmed reads were mapped to sea urchin Spur_v3.1 reference genome (containing the λ phage genome as chrLambda) 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 methylated 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
Supplementary_files_format_and_content: bedGraph / 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
|
| |
|
| Submission date |
Nov 06, 2021 |
| Last update date |
Nov 08, 2021 |
| Contact name |
Ksenia Skvortsova |
| E-mail(s) |
k.skvortsova@garvan.org.au
|
| Phone |
478135210
|
| Organization name |
Garvan Institute
|
| Department |
Genomics and Epigenetics
|
| Lab |
Developmental Epigenomics
|
| Street address |
384 Victoria St, Darlinghurst
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL30935 |
| Series (2) |
| GSE188333 |
Base-resolution DNA methylation maps of purple sea urchin (Strongylocentrotus purpuratus) |
| GSE188334 |
Base-resolution 5-hydroxymethylcytosine maps of sea urchin and lancelet embryos and adult tissues |
|
| Relations |
| BioSample |
SAMN22959048 |
| SRA |
SRX13025546 |
