 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 16, 2017 |
| Title |
8-Cell_embryo10_single_cell_1 |
| Sample type |
SRA |
| |
|
| Source name |
Mouse 8-cell blastomere
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 & 129sv
cell type: 8-cell embryo
passages / stage: 8-cell embryo
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The released genomic DNA were used to construct the PBAT library, as previously described (Guo et al., 2015; Smallwood et al., 2014).
Briefly, the single-cell genomic DNA, together with unmethylated lambda DNA (New England Biolabs) spike-ins, were subjected to bisulfite conversion using a MethylCode Bisulfite Conversion Kit (Invitrogen) according to the manufacturer’s instructions. The bisulfite converted DNAs were then annealed using random nonamer primers with a 5’- biotin tag and a truncated Illumina P5 adaptor (5’-biotin- CTACACGACGCTCTTCCGATCTNNNNNNNNN-3’) supplemented with 50 units of klenow polymerase (3’ to 5’ exo-, New England Biolabs). The excess primers were removed using 40 U exonuclease I (NEB) before DNA was purified using 0.8× Agencourt Ampure XP beads (Beckman Coulter). Then, the newly synthesized DNA strands were immobilized using the Dynabeads M280 Streptavidin (Invitrogen), and the original bisulfite-treated DNA templates were removed via two rounds of 0.1 N NaOH washes. The second strands were synthesized using 50 units klenow polymerase with random nonamer primers containing a truncated P7 Illumina adaptor (5’-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3’). The beads were further collected and washed several times, and the library was generated with 13 cycles of PCR amplifications using 1 U Kapa HiFi HS DNA Polymerase (Kapa Biosystems), together with 0.4 μM Illumina Forward PE1.0 primer (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and 0.4 μM pre-indexed Illumina Reverse primer (5’-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’; the underlined hexamer indicates the index sequences). Amplified libraries were purified with 0.8× Agencourt Ampure XP beads twice and were assessed on the Agilent Bioanalyzer 2100 platform and quantified with a standard curve-based qPCR assay (Kapa Biosystems). The final quality- ensured libraries were pooled and sequenced on the Illumina HiSeq2500 sequencer for 150 bp paired-end sequencing.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina CASAVA version 1.8 were used to the basecalling.
Raw reads were trimmed to remove the first 9 bases and remove the adapter-contaminated and low-quality reads using Trim Galore (v0.3.3).
The cleaned and QC-ensured reads were then aligned to the in silico bisulfite-converted mouse genome reference (mm9) using Bismark (v0.7.6) with paired-end and non-directional mapping parameters. After paired-end mapping, the unmapped reads were re-aligned to the same reference genome in single-end mode. Duplicated reads were excluded using SAMtools (v0.1.18) and then used for the subsequent analysis.
WCG and GCH methylation level of cytosines was estimated using comstomized scripts.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files which include the methylation level of cytosines in WCG and GCH context respectively
|
| |
|
| Submission date |
Apr 05, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Lin Li |
| E-mail(s) |
lilin2019@i.smu.edu.cn
|
| Organization name |
Southern Medical University
|
| Department |
Pathophysiology
|
| Street address |
Shatai South Road
|
| City |
Guangzhou |
| ZIP/Postal code |
510515 |
| Country |
China |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE78140 |
Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells |
|
| Relations |
| BioSample |
SAMN06688333 |
| SRA |
SRX2709427 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2564199_8-Cell_embryo10_single_cell_1.ACG.TCG.bw |
18.7 Mb |
(ftp)(http) |
BW |
| GSM2564199_8-Cell_embryo10_single_cell_1.GCA.GCC.GCT.bw |
131.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
