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Sample GSM1542461

Query DataSets for GSM1542461

Status

Public on Jul 13, 2016

Title

control hiPSC-CMs at 2 weeks #1

Sample type

SRA

Source name

cardiomyocytes

Organism

Homo sapiens

Characteristics

cell type: induced pluripotent stem cell derived cardiomyocytes
passages: 25-30
genotype: TBX20 wt/wt
cardiac phenotype: no cardaic disease

Treatment protocol

hiPSC-CMs were cultured in RPMI with B27 supplement and 2% FBS 2 days before experiment to accelerate the proliferation.

Growth protocol

hiPSCs were grown to 90% confluence and differentiated subsequently into beating cardiomyocytes using a small molecule-based monolayer method adapted after4 and described in detail previously.3 10 days after cardiac differentiation, hiPSC-CM monolayers were purified using non-glucose culture medium containing RPMI-1640 without glucose (Life technologies) and B27 supplement (Life technologies). Non-glucose culture medium was changed every two days and after 5 days, hiPSC-CMs were dissociated using TripleE (10 min), washed and plated onto matrigel-coated plates in culture medium with glucose.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted and quantified using miRNeasy Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s protocol. An additional DNAseI digestion step was performed to ensure that the samples were not contaminated with genomic DNA. Following the Qiagen RNA sample preparation protocol, 10 ug of total RNA was used to generate index-tagged paired-end cDNA libraries.5 Briefly, mRNAs were purified by polyA enrichment procedure using Dynal Oligo(dT) beads (Life technologies).
mRNA fragmentation was performed using RNA Fragmentation Reagents (Life technologies) with incubation at 70oC for 1 min to obtain 200-300b fragments. cDNA was generated using SuperScript Double-Stranded cDNA Synthesis Kit (Life technologies). Illumina sequencing adapters were ligated to cDNA using LigaFast (Promega) and PE Adapter Oligo Mix (Illumina, San Diego, CA). PCR was performed on the adapter-ligated cDNA with 2X Phusion DNA polymerase Master Mix (New England Biolabs, Ipswich, MA) using the following conditions: denaturation 98°C for 30 seconds, followed by 15 cycles of denaturation 98°C for 10 seconds, annealing 65°C for 30 seconds, and extension 72°C for 30 seconds, ending with an extension at 72°C for 5 min. After cDNA libraries were generated using the sample preparation kit, quality of the libraries was checked using Bioanalyzer (Agilent Technologies).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Paired-end fastq sequence reads from each sample were assembled against hg19 using the Tophat v2.0.6 with the Illumina-supplied hg19 gene-model annotation file ( gtf annotation).
The expression level (FPKM) was estimated by Cufflinks.
Cuffdiff was used to call differentially expressed genes with false discovery rate less than 0.05. Cuffdiff was run against the UCSC iGenomes GTF file from Illumina.
Subsequent to determining which genes were differentially expressed, FPKM was used for ﬁltering, and visualization purposes.
Genome_build: hg19

Submission date

Nov 10, 2014

Last update date

May 15, 2019

Contact name

Kazuki Kodo

E-mail(s)

kkodo@stanford.edu

Organization name

Stanford

Street address

campus street