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| Status |
Public on Feb 07, 2023 |
| Title |
scTrioseq2Met_5C_sc32 |
| Sample type |
SRA |
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| Source name |
Cells induced from SSC
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6[female] X DBA[male]
treatment: 5C
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| Treatment protocol |
The reprogramming process is induced with- or without five mechanicals in 24-well culture plates.
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| Growth protocol |
SSCs were cultured in StemPro-34 SFM (Gibico, USA) supplemented with 20 ng/ml EGF, 10 ng/ml bFGF, 10 ng/ml GDNFand 1000 U/ml ESGRO. gPSC were cultured in N2B27 medium supplemented with 2iL.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For modified STRT-seq and scTrio-seq2, RNA and DNA are extracted with lysis buffer. For PBAT and WGBS, genomic DNA was extracted using FastPure Cell/Tissue DNA Isolation Mini Kit.
The libraries for modified STRT-seq, Trio-seq2, PBAT and WGBS are performed according to the previously reported studies (Dong et al., 2018) (Bian et al., 2018) (Smallwood et al., 2014) (Li et al., 2010)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: Trio-seq2 (DNAme)
For scRNA-seq data, We firstly extracted cell barcodes and UMI sequences from read 2 of raw fastq data by UMItools (v1.0.0) (Smith et al., 2017) and attached them after the read name of read 1. After TSO and polyA sequences removal by local scripts, the “clean” reads were aligned to the mouse mm10 reference genome using STAR (2.7.2b) (Dobin et al., 2013), gene features were assigned by featureCounts (v2.0.0) (Liao et al., 2014). We used UMItools to generate UMI count expression matrix. For the quality control of scRNA-seq data, cells with detected gene numbers fewer than 2,000 or the fraction of mitochondrial reads more than 5% were excluded from further analysis, besides, genes expressed in fewer than 3 cells were filtered out for analysis.
For bisulfite sequencing data, trim_galore (v0.6.4) were used to remove low quality reads and adaptor sequence, and 9 bp of random primers from 5’ end of both read 1 and read 2 were also trimmed. Clean reads were then mapped to mm10 genome using Bismark (v0.23.0) with paired-end mode and ‘--non_directional’ option. Unmapped reads were outputted and mapped again by single-end mode. Aligned bam files were merged and sorted by samtools (v 1.11) and PCR duplicates were marked by picard MarkDuplicates (v2.23.6). Methylation calls for each CpG site were extracted by ‘bismark_methylation_extractor’ of Bismark. The thresholds for CpG sites coverage were 1X and 3X for single-cell- and bulk DNA methylation, respectively. Only methylation levels less than 10% or greater than 90% were retained for single-cell methylation data. Bisulfite conversion rate (BSCR) was estimated by methylation ratio of lambda DNA, samples with BSCR greater than 98% and detected CpG sites greater than 1,000,000 were kept for further analysis.
Genome_build: mm10
Supplementary_files_format_and_content: count matrix: matrix of UMI counts for all genes in each cell
Supplementary_files_format_and_content: bismark coverage: genome-wide cytosine report outputs of Bismark methylation extractor
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| Submission date |
Oct 20, 2021 |
| Last update date |
Feb 07, 2023 |
| Contact name |
Jiansen Lu |
| E-mail(s) |
jiansenlu@pku.edu.cn
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| Organization name |
Peking University
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| Department |
Biomedical Pioneering Innovation Center (BIOPIC)
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| Lab |
Fuchou Tang
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| Street address |
No. 5 Yiheyuan Road, Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE186260 |
The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting |
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| Relations |
| BioSample |
SAMN22442328 |
| SRA |
SRX12708123 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5642005_5C_Trio_DNAme_32.duprm.filtered.cov.gz |
5.6 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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