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| Status |
Public on Dec 06, 2017 |
| Title |
i12J-iPSC_RRBS-seq |
| Sample type |
SRA |
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| Source name |
iPSCs
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Pluripotent stem cells
passage: 20-25
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| Treatment protocol |
Different reprogramming mechanisms: iPSCs, somatic cell nuclear transfer, and IVF.
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| Growth protocol |
Pluripotent stem cells (iPSC, nt-ESCs, and IVF-ESCs) were maintained in chemically defined E8 medium on Matrigel-coated plates. PSC-derived cardiomyocytes (PSC-CMs) were cultured in RPMI-1640 medium plus B27 supplement on Matrigel-coated plates. PSC-derived endothelial cells (PSC-ECs) were maintained in EGM2 medium on 0.2% gelatin-coated plates.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNAs were extracted by RNeasy Plus Mini kit (Qiagen, cat no. 74134) according to the manufacturer’s protocol. Genomic DNA was isolated using a DNeasy Blood and Tissue kit (Qiagen, cat no. 69506).
For RNA-seq library preparation, mRNAs were isolated from total RNAs using the Dynabeads Oligo (dT)25 in a Dynabeads mRNA DIRECT Micro Kit (Thermo Fisher Scientific, cat no. 61021). ERCC Spike-In Control Mix (Thermo Fisher Scientific, cat no. 4456740) was added to total RNA input before mRNA isolation. RNA-seq libraries were constructed using an Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific, cat no. 4479789). For RRBS-seq library construction, 300 ng of genomic DNA was digested with MspI (NEB, cat no. R0106S) at 37°C for 1 hr. Unmethylated lambda DNA (Promega, cat no. D1521) was spiked in as the quality control for sodium bisulfite conversion. The DNA fragments were then purified and selected using Agencourt AMPure XP beads (Beckman Coulter, cat no. A63880). DNA fragments were repaired to blunt ends by Klenow fragments (NEB, cat no. M0212S). DNA fragments were then ligated to the methylated Illumina adapters (NEB, cat no. E7535L) using a NEBNext Ultra Ligation Module (NEB, cat no. E7445S). The ligation product was purified using Agencourt AMPure XP beads and subsequently subjected to sodium bisulfite conversion using an EZ DNA Methylation-Gold Kit (Zymo Research, cat no. D5005). The purified bisulfite-converted DNA was amplified using the EpiMark Hot Start Taq DNA Polymerase (NEB, cat no. M0490). NEBNext Index Primers were used to barcode different samples during PCR amplification.
RNA-seq and RRBS-seq
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
hisat2 -x hg38.idx -U ESO7-CM_rep1.fastq.gz -S ESO7-CM_rep1.sam -p 2
featureCounts -T 2 -t exon -g gene_name -s 1 -a hg38_P2_ERCC.gtf -o MingTao_NT_mRNA.txt 12C-CM_rep1_sorted.bam 12C-CM_rep2_sorted.bam 12C-EC_rep1_sorted.bam
trim_galore --paired ESO7-CM_R1.fq.gz ESO7-CM_R2.fq.gz
bismark bt2_hg38 --bowtie2 -1 ESO7-CM_R1_val_1.fq.gz -2 ESO7-CM_R2_val_2.fq.gz
MethylKit
Genome_build: hg38
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| Submission date |
Jan 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph Wu |
| Organization name |
Stanford University School of Medicine
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| Street address |
Stanford University School of Medicine
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| City |
Stanford |
| State/province |
Carlifornia |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE94267 |
Molecular and Functional Resemblance of Terminally Differentiated Cells Derived from Isogenic Human iPSCs and Somatic Cell Nuclear Transfer Derived ESCs |
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| Relations |
| BioSample |
SAMN06279669 |
| SRA |
SRX2527532 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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