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| Status |
Public on Jul 27, 2020 |
| Title |
PBAT_EpiLC_dCD |
| Sample type |
SRA |
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| Source name |
epiblast like cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: d2 EpiLC
strain: R1
genotype: Mll3/4 dCD
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| Growth protocol |
ESC were cultured on gelatin-coated plates using Knock-out DMEM supplemented with 15% FBS and LIF. Before each PGCLC differentiation, ESC were adapted to 2i+LIF media (serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 [0.4 μM], GSK3β inhibitor CHIR99021 [3 μM], and LIF) for at least four days in gelatin-coated tissue culture plates. For the EpiLC differentiation, 6-well plates were coated with 15 µg ml-1 Fibronectin and 2 x 105 2i ESC were differentiated in N2B27 media supplemented with 20 ng ml-1 Activin A, 12 ng ml-1 bFGF and 10 µl ml-1 KSR for two days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Bisulfite sequencing libraries were prepared from column-purified DNA of d2 EpiLC using the PBAT method previously described in (Smallwood et al., 2014). Briefly, for the bisulfite conversion the instructions of the EZ Methylation Direct MagPrep Kit (Zymo) were followed.
After purification, bisulfite converted DNAs were eluted from MagBeads directly into 39 µl of first strand synthesis reaction mastermix (1x Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 µM 6NF preamp oligo (IDT)), heated to 65 °C for 3 minutes and cooled on ice. 50 U of klenow exo- (Enzymatics) was added and the mixture incubated on a thermocycler at 37 °C for 30 minutes after slowly ramping from 4 °C. Reactions were diluted to 100 ml and 20 U of exonuclease I (NEB) added and incubated at 37 °C before purification using a 0.8:1 ratio of AMPure XP beads. Purified products were resuspended in 50 ml of second strand mastermix (1x Blue Buffer (Enzymatics), 0.4 mM dNTP mix (Roche), 0.4 µM 6NR adaptor 2 oligo (IDT) then heated to 98 °C for 2 minutes and cooled on ice. 50 U of klenow exo- (Enzymatics) was added and the mixture incubated on a thermocycler at 37 °C for 90 minutes after slowly ramping from 4 °C. Second strand products were purified using a 0.8:1 ratio of AMPure XP beads and resuspended in 50 ml of PCR mastermix (1x KAPA HiFi Readymix, 0.2 µM PE1.0 primer, 0.2 µM iTAG index primer) and amplified with 12 cycles. The final libraries were purified using a 0.8:1 volumetric ratio of AMPure XP beads before pooling and sequencing. All libraries were prepared in parallel with the pre-PCR purification steps carried out using a Bravo Workstation pipetting robot (Agilent Technologies).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Genome-wide DNA methylation data was analysed with Bismark (Krueger and Andrews 2011). Briefly, adapter trimming was performed with Trim Galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with trim_galore --clip_R1 6 —clip_R2 6 --paired. The resulting reads were mapped with Bismark-v0.16.1 (Krueger and Andrews 2011) and bowtie2-2.2.9 (Langmead and Salzberg 2012), using the “-pbat” setting. Additionally the unmapped reads were remapped as single-end reads and the CpG methylation levels were estimated with the Bismark methylation extractor. Finally only CpGs with a coverage of 3 - 100 reads were considered.
Genome_build: mm10
Supplementary_files_format_and_content: CpG methylation coverage
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| Submission date |
Jul 24, 2020 |
| Last update date |
Jan 29, 2021 |
| Contact name |
Alvaro Rada-Iglesias |
| E-mail(s) |
alvaro.rada@unican.es
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| Organization name |
Institute of Biomedicine and Biotechnology of Cantabria (IBBTEC), CSIC/University of Cantabria
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| Street address |
Albert Einstein 22, PCTCAN
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| City |
Santander |
| State/province |
Cantabria |
| ZIP/Postal code |
39011 |
| Country |
Spain |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE155083 |
Enhancer-associated H3K4 methylation safeguards in vitro germline competence [WGBS] |
| GSE155089 |
Enhancer-associated H3K4 methylation safeguards in vitro germline competence |
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| Relations |
| BioSample |
SAMN15639661 |
| SRA |
SRX8819579 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4694891_WGBS_EpiLC_Mll34_dCD.cov.gz |
169.8 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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