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| Status |
Public on May 07, 2021 |
| Title |
DNA_Granulosa_sc32 |
| Sample type |
SRA |
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| Source name |
Ovarian
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Human somatic cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the isolation of human growing oocytes and ovarian somatic cells, fresh ovarian tissue (~1cm3) was cut into pieces after the blood vessels and cysts were removed. Then, pieces of ovarian tissue were dissociated in 2ml of 1× DPBS (Gibco) with 5% collagenase (Gibco) at 37°C for 1 h, 1000× rpm on a thermo incubator. After the first round dissociation, the sample was filtrated through a 100-µm cell strainer (Biololgix Group). Single cells were centrifuged at 300× g for 5 min and resuspended in 2ml LFP medium (1× L15 medium (Gibco), 10% FBS (Vistec) and 1% penicillin–streptomycin (Gibco)) before picking up. Next, the undissociated tissue sample underwent the second round digestion by 2ml TrypLE™ Express (Gibco) at 37°C for 30 min, 1000× rpm on a thermo incubator. Then the digested sample was filtrated through a 100-µm cell strainer (Biololgix Group). Finally, single cells were centrifuged at 300× g for 5 min and resuspended in 2ml LFP medium before selection. Individual cell was picked and subjected to the scChaRM-seq steps. For mature human oocytes, zona pellucida was removed by Tyrode's Solution (Sigma). Then, oocytes were washed in 1× DPBS with 1% BSA by several times under a stereomicroscope. Individual oocyte was picked and subjected to the scChaRM-seq steps.
For scChaRM-seq, single cells were individually picked into 0.25-ml PCR tubes containing 1.825 µl of ice-cold lysis buffer. Then, in vitro GpC methylation was performed by adding 2.5 U of M.CviPI (New England Biolabs) and 160 μM SAM (New England Biolabs)directly to the single-cell lysate. Samples were incubated at 37°C for 15 min, and the reaction was stopped by adding 5 μl of RLT buffer (Qiagen). Single-cell mRNA was captured by biotin-tagged and barcoded-oligo-dT primer conjugated to magnetic streptavidin beads (Invitrogen), and the gDNA supernatant was transferred into another PCR tube for methylome analysis. The mRNA separated from single cells was subjected to cDNA synthesis, PCR amplification and library construction according to our previously reported protocol. The corresponding gDNA from single cells was first purified by AMPureXP beads (Beckman Coulter), then subjected to bisulfite conversion (Zymo Research) and DNA library construction by our recently published separated (TAILS) method. For SCRB-seq, single cells were picked, lysed and subjected to first-strand cDNA synthesis as reported previously. Then, second-strand cDNA was synthesized, amplified and fragmented. An RNA-seq library was prepared by following the instruction manual of the NEBNext UltraII DNA Library Prep Kit for Illumina (New England Biolabs). Finally, the libraries were checked, pooled and sequenced with paired-end 150-bp reads on the Illumina HiSeq X-Ten platform (Novogene).
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
HiSeq X Ten |
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| Description |
Bisulfite-sequencing data of single human somatic cells profiled by scChaRM-seq
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| Data processing |
Processing of the scChaRM-seq DNA data: Trim Galore (v0.4.4) was used to remove random primer sequences, low-quality bases and adaptor sequences from the single-cell bisulfite data. Only reads longer than 50 bp after trimming were retained for alignment. Then, clean reads were mapped to the human reference genome hg19 by Bismark (v0.7.6) in paired-end mode, and unmapped reads were re-aligned to the reference genome in single-end mode. Duplicate reads from PCR were removed by the SAMtools (v0.1.18) command “samtools rmdup”, and only nonduplicate reads were used to perform the downstream analysis. The GCH (G=A, T or C) and WCG (W=A or T) sites (≥1× sequencing depth) in each single cell were summed. Then, the chromatin accessibility and DNA methylation level were calculated as the average GCH or WCG level, respectively.
Processing of the single-cell RNA-seq data (including data from scChaRM-seq and SCRB-seq): First, raw reads were trimmed by Cutadapt (v1.15). Clean reads for each individual cell were then classified according to cell barcodes on Read#2. Then, Read#1 was aligned to the hg19 human transcriptome by STAR (v2.6.0a) with default parameters. The transcript copy number of each gene was counted by HTseq (v0.10.0) after de-duplication based on UMIs. Cells with fewer than 10,000 transcripts or with fewer than 1,000 detected genes were excluded. Genes with an average count less than 0.1 among all retained cells were filtered out.
Genome_build: hg19
Supplementary_files_format_and_content: *bw: bigwig files (including aggregated WCG and GCH sites of eight cell types); hO_scChaRM_count_matix.xls.gz: Count matrix in tab-separated value files; barcodes_and_cell_information.xlsx.gz: Excel data file including sequences of barcodes used and annotations of cells processed by scChaRM-seq.
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| Submission date |
Jul 20, 2020 |
| Last update date |
May 07, 2021 |
| Contact name |
Fan Guo |
| E-mail(s) |
[email protected]
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| Organization name |
Institute of Zoology, Chinese Academy of Sciences
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| Department |
The State Key Laboratory of Stem Cell and Reproductive Biology
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| Lab |
Group of Reproductive Epigenetics
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| Street address |
1 Beichen West Road, Chaoyang District
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| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE154762 |
Decoding dynamic epigenetic landscapes in human oocytes using single-cell multi-omics sequencing |
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| Relations |
| BioSample |
SAMN15585061 |
| SRA |
SRX8775705 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4678798_DNA_Granulosa_sc32.GCH.bw |
13.2 Mb |
(ftp)(http) |
BW |
| GSM4678798_DNA_Granulosa_sc32.WCG.bw |
1.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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