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| Status |
Public on Dec 01, 2016 |
| Title |
pr9-iPSC.H3K27me3.ChIP-Seq |
| Sample type |
SRA |
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| Source name |
primed iPSCs
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| Organism |
Homo sapiens |
| Characteristics |
subject status: β-thalassemia patients carrying the β-41/42 mutation
cell type: Fibroblasts
cell subtype: conventional iPSCs
chip antibody: H3K27me3
chip antibody vendor: Millipore
chip antibody cat. #: 07-449
chip antibody lot #: JBC1873477
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| Growth protocol |
Fibroblasts were isolated from β-thalassemia patients carrying the β-41/42 mutation. Episomal vectors carrying a combination of transcription factors encoded by Oct4, Sox2, Klf4 and c-Myc were transfected into fibroblasts through electroporation and were then cultured in conventional hESM containing DMEM/F12 (Invitrogen) supplemented with 20% Knockout Serum Replacement (Invitrogen), 10 ng/ml bFGF (Peprotech), 10-4M non-essential amino acids (Millipore), 10-4M β-mercaptoethanol (Millipore), 2 mM L-Glutamax (Invitrogen) and 50 µg/ml penicillin/streptomycin (Millipore) for 6 days. Then, the medium was replaced with human naïve medium (5i/L/FA medium) and cultured for 14-20 days. Dome-shaped colonies similar to mouse ESCs were selected and expanded by single cell passaging.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA-Seq, total RNA was isolated from naïve iPSC lines and primed PSC lines using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA sequencing libraries were constructed from the extracted RNA using standard Illumina libraries prep protocols. Sequencing was performed on Illumina HiSeq2000 platform in pair end.
For ChIP-Seq, Cells (~1x10^7) for each sample were fixed in 1% formaldehyde for 10 min. The crosslink reaction was stopped by addition of glycine. Next, cells were washed 3 times with PBS. Then, cells were resuspended in lysis buffer and sonicated in Diagenode Bioruptor (Biosence) to fragments with length range of 200-500bp. Antibodies to H3K4me3 and H3K27me3 (Abcam) were added to supernatants to capture nucleosomes with these two histone modifications, respectively. The nucleosomal DNA was recovered by removal of histones at overnight incubation at 65℃ and purified using QIAquick PCR purification kit (QIAGEN, Maryland). The ChIP’ed DNA was used to construct libraries for sequencing using the ChIP-seq Sample Prep Kit (Illumina) according manufacturer’s instructions.
The RNA sequencing libraries were constructed from the extracted RNA using standard Illumina libraries prep protocols. The ChIP’ed DNA was used to construct libraries for sequencing using the ChIP-seq Sample Prep Kit (Illumina) according manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
RNA-seq reads were aligned to the UCSC human genes (hg19) using Tophat (version 2.0.13). Only the uniquely mapped reads in proper pairs were retained for analysis in this study.
We used Cuffdiff (version 2.1.1) to calculate the expression abundance of each gene as fragments per kilobase per million mapped reads (FPKM).
ChIP-Seq reads were aligned to the human reference genome (hg19) using Bowtie (version 2.1.0). Only the uniquely mapped reads in proper pairs were retained for analysis in this study.
We used the MACS (version 1.4.1) peak finding algorithm to detect regions of ChIP-Seq signal enrichment (p-value < 1e-5) over background.
Genome_build: hg19
Supplementary_files_format_and_content: For ChIP-Seq data, processed data files are tab-delimited text files with enriched peaks predicted by MACS. For RNA-Seq data, processed data files are tab-delimited files showing gene expresssion levels with FPKM.
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| Submission date |
May 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Xiaobai Zhang |
| E-mail(s) |
[email protected]
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| Organization name |
Tongji University
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| Street address |
1239 Siping Road
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| City |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
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| Platform ID |
GPL9052 |
| Series (1) |
| GSE69319 |
Naïve iPSCs Generated from β-thalassemia Fibroblasts Allow Efficient Gene Correction with CRISPR/Cas9 |
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| Relations |
| BioSample |
SAMN03741930 |
| SRA |
SRX1041548 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1697675_pr9-iPSC.H3K27me3.peak.txt.gz |
203.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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