 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 28, 2023 |
| Title |
10_XT |
| Sample type |
SRA |
| |
|
| Source name |
hindlimb webbing
|
| Organism |
Xenopus tropicalis |
| Characteristics |
tissue: hindlimb webbing
strain: sox3 F0
dob: 25/07/2018
|
| Treatment protocol |
Frogs were kept in tanks with constant temperature of 25°C and 12/12 hour light cycle.
|
| Growth protocol |
Sixteen adult Xenopus tropicalis were housed at the National Xenopus Resource (RRID:SCR_013731) in two multi-rack recirculating aquatic systems with established diet and water parameters (conductivity, pH, and temperature) as previously defined.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Disposable biopsy punches were used to collect tissue (VWR 21909–140) from the hindlimb webbing of the frogs. DNA extraction was performed from web punches (4 mm in diameter except 6-months old for which we did 2mm), according to Xu Y. et al. (Xu et al. 2020) with minor modifications. Briefly, tissue chunks were digested o/n in a 1.5 ml tube containing 200 µl of lysis buffer (100 mM Tris-HCl pH 8; 200 mM NaCl; 0.20% SDS; 5 mM EDTA) + 4 µl of Proteinase K (20 mg/ml, NEB) at 55°C (300 rpm continuous shaking in a thermomixer). After centrifugation for 15 min at 16'000g (room temperature), the supernatant was transferred into a new tube avoiding the debris. The centrifugation step was repeated after the addition and mixing of the same volume of isopropanol. The resulting pellet was then washed twice with 500 µl of EtOH 70% (centrifugation at 16'000g for 10 min, RT). The remaining ethanol was then carefully removed and the pellet air dried for 5-10 minutes at 55°C (open caps). The dried pellet was then resuspended with 55 µl of EB buffer (10 mM Tris-HCl pH 8) at 55°C for 1 h at 1400 rpm in a thermomixer, before being quantified (Qubit dsDNA BR - Life Technologies) and quality checked (Agilent 4200 TapeStation - Genomic Assay).
500 ng of purified DNA was sonicated using the Bioruptor Pico (Diagenode) for 15 cycles of 30 second ON / 90 seconds OFF. Libraries were prepared according to Morselli et al. (Morselli et al. 2022) with minor modifications. Briefly, NEB Next Ultra II DNA kit was used for end-repair, A-tailing and ligation of pre-methylated unique-dual indexed adapters (Morselli et al., Methods 2021). Hybridization capture was performed as described in Morselli et al., Epigenetics 2022). Bisulfite conversion was performed with EZ DNA Methylation-Gold (Zymo Research) according to the manufacturer's instructions. The final amplification was performed with KAPA HiFi U+ (Roche Sequencing), IDT xGen Primers (20 µM - Integrated DNA Technologies), and 12 PCR cycles. Library QC was performed using the D1000 Assay on a 4200 Agilent TapeStation, and its concentration measured with the Qubit dsDNA BR Assay (Life Technologies). Libraries were sequenced on a NovaSeq6000 (SP lane) as paired-end 150 bases.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Xen-capture1
Targeted Bisulfite Sequencing (TBSeq)
|
| Data processing |
Demultiplexed Fastq files were subject to QC (FastQC - Babraham Bioinformatics) and trimming with cutadapt v2.10 (Martin 2011) (options: -u -10 -U 10 -q 20 -m 50).
Alignment to the Xenopus tropicalis genome (version XENTR_10.0) with BSBolt Align (Farrell et al. 2021) (default options).
PCR duplicates were removed with samtools markdup (option -r) v1.15 (Danecek et al. 2021).
DNA methylation was called using BSBolt CallMethylation v1.3.0 (options: -BQ 10 -MQ 20 -IO) resulting in CGmap files.
The matrices of common CpG sites (with at least 100x coverage) were produced using BSBolt AggregateMatrix.
Assembly: Xenopus tropicalis genome (version XENTR_10.0)
Supplementary files format and content: Xen-CGmatrix_TBSeqPilot_100x_100.txt: Tab-delimited text file, CGmatrix. First column = chromosome:position; from the second column = methylation value of common cytosines (CpG context) covered by at least 3 reads. One column for each sample.
Library strategy: TBSeq
|
| |
|
| Submission date |
Jan 03, 2023 |
| Last update date |
Apr 28, 2023 |
| Contact name |
Marco Morselli |
| E-mail(s) |
marco.morselli@unipr.it
|
| Organization name |
University of Parma
|
| Department |
S.C.V.S.A.
|
| Street address |
Parco Area delle Scienze, 23/A
|
| City |
Parma |
| State/province |
Parma |
| ZIP/Postal code |
43124 |
| Country |
Italy |
| |
|
| Platform ID |
GPL30018 |
| Series (2) |
| GSE222107 |
Targeted bisulfite sequencing to profile age-associated DNA methylation changes in Xenopus frogs |
| GSE222108 |
Age-associated DNA methylation changes in Xenopus frogs, and Targeted bisulfite sequencing to profile age-associated DNA methylation changes in Xenopus frogs |
|
| Relations |
| BioSample |
SAMN32549140 |
| SRA |
SRX18920355 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
