Dataset View [GSE63818]

TitleThe Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells
ArticleQiao J,Tang F,Gao YQ,Wen L,Zhu X,Ren Y,Liu S,Zhang L,Li J,Zhu P,Hou Y,Zhang W,Jin H,Zhang Y,Zhi X,Yan J,Li R,Wang W,Wei Y,Wang X,Hu Y,Yong J,Zhao Y,Hu B,Li L,Guo H,Yan L,Guo F.The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells.Cell.2015 Jun 4
Bio ProjectBioProject: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA269154
SraSRA: http://www.ncbi.nlm.nih.gov/sra?term=SRP050499
Overall DesginIn total, we analyzed the transcriptomes of 233 individual male and female human PGCs from 15 embryos at between 4 and 19 weeks of gestation as well as 86 neighboring somatic cells from 13 of these embryos using a single-cell RNA-Seq method that we developed. Furthermore, we analyzed the DNA methylomes of both male and female human gonadal PGCs as well as the neighboring somatic cells of eleven of these embryos at between 7 and 19 weeks of gestation using whole-genome bisulfite sequencing (WGBS). In total, we generated 1.3 billion 100 bp paired-end reads for transcriptome analyses and 4.1 billion 100 bp or 150 bp paired-end reads for DNA methylome analyses.
SummaryGerm cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10-11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation; with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.
Experimental ProtocolFor single cell transcriptome analysis, the protocol for single-cell RNA-Seq has been published previously (Tang et al., 2010; Tang et al., 2009). Briefly, after a MACS or FACS procedure, a single PGC or somatic cell was randomly picked by using a mouth pipette and transferred into lysate buffer. And for DNA methylome analysis, the KIT-positive PGCs that were obtained by FACS were further washed with DPBS, and then DNA was isolated from the cell pellets using DNeasy Blood & Tissue Kits (Qiagen).; For single cell RNA-seq library construction, a total of 20–100 ng of cDNA was sheared into 150 bp to 350 bp by Covaris S2 after the generation of cDNA from a single cell, and the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Inc.) was used to prepare the sequencing library following the manufacturer’s protocol. Briefly, the fragmented cDNA was end-repaired, dA tailed, adapter ligated and then subjected to 10–12 cycles of PCR amplification. Libraries were pooled and sequenced on Illumina HiSeq2500 sequencers for 100-bp paired-end sequencing. And for whole genome bisulfite sequencing (WGBS) library construction, samples of 5–100 ng of genomic DNA were used to construct the PBAT library, as previously described with minor modifications (Miura et al., 2012; Smallwood et al., 2014). Briefly, the isolated genomic DNAs, together with 1% unmethylated lambda DNA (Thermo Scientific), were subjected to bisulfite conversion using a MethylCode Bisulfite Conversion Kit (Invitrogen) according to the manufacturer’s instructions. The bisulfite-converted templates were then annealed using random nonamer primers with a 5’ biotin tag and a truncated Illumina P5 adapter (5’-biotin- CTACACGACGCTCTTCCGATCTNNNNNNNNN-3’) supplemented with 50 units of klenow polymerase (3’ to 5’ exo-, NEB). 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 (3’ to 5’ exo-, NEB) with random nonamer primers containing a truncated P7 Illumina adapter (5’-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3’). The beads were further collected and were washed several times, and the library was finally amplified with 4–6 cycles of PCR 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 Reversed primer (5’-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’; the underlined hexamer indicates 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 HiSeq2000/2500 sequencer for 100 bp or 150 bp paired-end sequencing.
Data processingIllumina CASAVA version 1.8 were used to the basecalling.; Reads were trimmed to remove the adapter sequences and low quality bases.; For RNA-seq, the cleaned reads were aligned to the human hg19 reference using Tophat (v2.0.12) with default settings (Trapnell et al., 2009). And for WGBS, bisulfite-converted reads were aligned to the human refference genome (hg19) using Bismark software (v0.7.6).; For RNA-seq, Cufflinks (v2.2.1) with default parameters was further used to assemble the transcripts and quantify transcription levels (FPKM, fragments per kilobase of transcript per million mapped reads) of annotated genes (Trapnell et al., 2010). And for WGBS, CpG methylation level of covered cytosines was estimated using comstomized Perl scripts.; Genome_build: hg19; Supplementary_files_format_and_content: txt files which include the FPKM values of RefSeq genes, and the bed files which include the methylation level of cytosines in CpG, CHH and CHG context.
Public OnPublic on Jun 03 2015

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