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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 16, 2017 |
| Title |
ES_10cells_3 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cell
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 & 129sv
cell type: Embryonic stem cell
passages / stage: 20-30
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| 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). Single-cell RNA-seq was performed under standard Smart-seq2 protocol. Bulk cells RNA-seq were performed under the instruction of mRNA library construction kit (NEB).
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.
Bisulfite-Seq and RNA-Seq
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Illumina CASAVA version 1.8 were used to the basecalling.
For COOL-Seq, 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). And for RNA-seq, reads were trimmed to remove the adapter and low quality bases with customized scripts.
For COOL-Seq, the cleaned 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. And for RNA-seq, the cleaned reads were aligned to the mouse reference (mm9) using Tophat (v2.0.12) with default settings (Trapnell et al., 2009).
WCG and GCH methylation level of cytosines was estimated using customized scripts.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files which include the methylation level of cytosines in WCG and GCH context respectively, and txt files which include the RPKM (reads per kilobase transcriptome per million reads) values of RefSeq genes.
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| Submission date |
Apr 10, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Lin Li |
| E-mail(s) |
lilin2019@i.smu.edu.cn
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| Organization name |
Southern Medical University
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| Department |
Pathophysiology
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| Street address |
Shatai South Road
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| City |
Guangzhou |
| ZIP/Postal code |
510515 |
| Country |
China |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE78140 |
Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells |
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| Relations |
| BioSample |
SAMN06706303 |
| SRA |
SRX2732216 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2573071_ES_10cells_3.ACG.TCG.bw |
36.4 Mb |
(ftp)(http) |
BW |
| GSM2573071_ES_10cells_3.GCA.GCC.GCT.bw |
266.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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