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Sample GSM7980824

Query DataSets for GSM7980824

Status

Public on Mar 15, 2024

Title

Bisulfite-seq_8C_CB_Female_rep1

Sample type

SRA

Source name

Single preimplantation embryo

Organisms

Mus musculus; Mus musculus castaneus

Characteristics

tissue: Single preimplantation embryo
cell type: N/A
genotype: wild type (MC)
Sex: Female
strain: C57BL/6J x CAST/EiJ
developmental stage: 8-cell
treatment: N/A

Extracted molecule

genomic DNA

Extraction protocol

Single embryo was lysed in RLT plus buffer (Qiagen).
Bisulfite DNAseq libraries were prepared following a previously published protocol, with some minimal modifications. Briefly, single embryo was lysed in RLT plus buffer (Qiagen), followed by the addition of unmethylated lambda DNA control. Genomic DNAs were treated with CT conversion reagent (EZ methylation kit, Zyon). After the purification using PureLink PCR Purifiation kit (ThermoFisher Scientific), DNAs were amplified 5 times using Preamp oligos and Klenow exo- polymerase (NEB). Amplified DNAs were depleted of single strand DNAs by adding exonuclease I, followed by the purification using Ampure XP beads. The second round of DNA synthesis reaction was carried out using Adapter 2 Oligos. Finally, all DNAs were purified and further amplified using 14 cycles of PCR. The yielded DNAs were purified and ready for bisulfite sequencing.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina HiSeq 2500

Data processing

On the HiSeq 2500 platform, intensity extraction from images, base calling and quality score assignment during the sequencing was performed by software RTA.
Additional adapters at the 3’ end and first 6 nucleotide bases from the 5’ end were trimmed from the raw reads, followed by the PCR duplicates removal.
The initial alignments of Bisulfite DNAseq data were carried out as previously described (Clark, S. J. et al., 2017). The processed reads were then aligned to both mm10 and pesudo-CAST genome using Bismark with the following parameters (--non_directional).
Due to the nature of bisulfite sequencing, CAST/EiJ SNPs including C to T, T to C, G to A and A to G were excluded from downstream allelic analysis to avoid false positive allelic decisions.
mm10
processed data files format and content: .bw files showing the rate of DNA methylation

Submission date

Dec 18, 2023

Last update date

Mar 15, 2024

Contact name

Chunyao Wei

E-mail(s)

weic@molbio.mgh.harvard.edu

Organization name

Massachusetts General Hospital

Department

Molecular Biology