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| Status |
Public on Aug 11, 2020 |
| Title |
ED_hiPSC-CM_2_RNA-seq |
| Sample type |
SRA |
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| Source name |
hiPSC-CM (Enhancer Deletion)
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| Organism |
Homo sapiens |
| Characteristics |
cell type: hiPSC-CM
gender: male
differentiation days: Day 20-31
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| Treatment protocol |
Cardiomyocytes were differentiated from human and NHP pluripotent cells. Briefly, differentiation medium consisted of RPMI-1640 media (11875-085, Life Technologies) supplemented with B27® minus insulin (A1895601, Life Technologies) (RPMI + B27-). To this medium, various small molecules were added over a week-long timetable as previously described. On the first day (D0) of differentiation, 6 µM CHIR 99021 (C-6556, LC Laboratories) was added. On D2, media was aspirated and replaced with RPMI + B27-. On D3, media was aspirated and replaced with 5 µM of IWR-1 (S7086, Selleck Chemicals) in RPMI + B27-. Media was replaced with RPMI + B27- on D5 and RPMI plus B27 supplemented with insulin (17504-044, Life Technologies) (RPMI + B27+) on D7.
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| Growth protocol |
Pluripotent cells were grown on Matrigel-coated plates (1:100 Matrigel in E8 medium) using chemically defined E8 medium. The medium was changed daily and cells were passaged every 3-4 days using EDTA or Accutase. Cardiomyocytes were maintained in RPMI + B27+ with media change every other day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq, RNA was isolated using miRNeasy kit (Qiagen) following the manufacture’s protocol. For ATAC-seq, 100,000 cells were centrifuged 500 g for 5 min at room temperature. The cell pellet was resuspended in 50 ml lysis buffer (10 mM Tris–Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Igepal CA-630) and centrifuged immediately 500 g for 10 min at 4°C. The cell pellet was resuspended in 50 ml transposase mixture (25 ul 2×TD buffer, 22.5 μl dH2O, and 2.5 μl Illumina Tn5 transposase or with final concentration of 100 nM Atto-590-labeled in-house Tn5) and incubated at 37°C for 30 min. After transposition, the mixture was purified with the Qiagen Mini-purification kit and eluted in 10 ul Qiagen EB elution buffer. For transgenic BANCR embryos plasmid vector construction (pRP[Exp]-BetaMHC>hBANCR) and purification, pronuclear plasmid injection of FVB embryos, harvesting and genotyping of E16 embryos were performed by Cyagen Biosciences (Santa Clara, CA,USA). Total RNA was isolated from extracted embryo hearts that were plasmid positive (n=2) or negative (n=2).
Total RNA was submitted to a core facility (Stanford Functional Genomics) for library preparation and Illumina sequencing. For ATAC-seq, sequencing libraries were prepared following the original ATAC-seq protocol (Buenrostro, J. D. et al. Nat Methods 10, 1213-1218 (2013)). The HiChIP protocol was performed as previously described (Mumbach, M. R. et al. Nat Methods 13, 919-922 (2016)).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
K6
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| Data processing |
ATAC-Seq reads alignment using Bowtie v2.2.6
Sort/remove duplicates using Sambamba v0.6.5
Convert to bam using Samtools v1.2.0
Count feature coverage around features using Deeptools v2.0.0
Count feature coverages using Rsubread v1.26.1
Trim reads/ quality control using Trim Galore v0.4.2
RNA-Seq reads alignment using STAR v2.5.3a
Count feature coverage around transcriptomic features using fetureCounts v1.5.0
Count feature coverages using igvtools v2.3.91
Detect differential regions using DESeq2 v1.16.1
ChIP-seq peaks called using MACS2 version 2.1.1
Genome_build: hg19/panTro4/gorGor3/rheMac3/mm9
Supplementary_files_format_and_content: TDF and BW format, read coverage
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| Submission date |
Mar 05, 2019 |
| Last update date |
Aug 12, 2020 |
| Contact name |
Lei Tian |
| E-mail(s) |
[email protected]
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| Phone |
6502388262
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| Organization name |
Stanford's Postdoctoral Scholar programs
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| Street address |
1215 Welch Rd
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (1) |
| GSE111930 |
An endogenous retrovirus-derived long non-coding RNA promotes fetal cardiomyocyte migration in primates |
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| Relations |
| BioSample |
SAMN11054961 |
| SRA |
SRX5464935 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3639701_ED_hiPSC-CM_2_RNA-seq.tdf |
81.5 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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