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Sample GSM3762945

Query DataSets for GSM3762945

Status

Public on Jun 15, 2019

Title

Mutant RAF1 S257L/+ rep2

Sample type

SRA

Source name

iPSC-derived cardiomyocytes

Organism

Homo sapiens

Characteristics

cell type: iPSC-derived cardiomyocytes
age: Day 24

Treatment protocol

At Day 22, mutant RAF1 cardiomyocytes were treated with 25 µM BIX02189 or 25 µM PD98059 for 48 hours before total RNA extraction

Growth protocol

iPSC-derived cardiomyocytes were differentiated using small molecules that modulate the Wnt-beta catenin pathway. At day 15 of differentiation, the cardiomyocytes were purified for 4 days with DL-Lactate supplementation. At Day 21, cardiomyocytes were replated on 0.1% gelatin coated plates and cultured in RPMI+10% FBS. At day 24, Total RNA was extracted

Extracted molecule

total RNA

Extraction protocol

Total RNA was prepared using the mirVana PARIS purification Kit (ThermoFisher, #AM1556) following the manufacturer’s recommendations. RNA integrity (RIN) was determined using ScreenTape R6K kit on the TapeStation2200 (Agilent Technologies) and only samples with RIN >7 were further used for RNA-sequencing
Isolated total RNA was diluted to 0.95 ng/µl and 10.5 µL was loaded in each reverse transcription reaction with the SuperScript VILO cDNA Synthesis Kit (ThermoFisher, #11754050). Reverse transcription was completed in a thermocycler with the following temperature settings: 42ºC for 30 minutes, 85ºC for 5 minutes and 10ºC on hold. After reverse transcription, libraries were prepared using the Ion Chef (ThermoFisher, #4484177) and the Ion AmpliSeq Transcriptome Human Gene Expression Panel, Chef-Ready Kit (ThermoFisher, #A31446). The AmpliSeq Gene Expression Panel includes one primer pair for every gene in the transcriptome, allowing for amplification of a single amplicon for each gene to be sequenced. At the completion of library preparation, libraries from 8 samples were pooled together, templated, and loaded on the Ion 540 Chip (ThermoFisher, #A27765) using the Ion Chef and the Ion 540 Kit Chef (ThermoFisher, #A27759). After templated samples were loaded onto the 540 Chips, each Chip was inserted into the Ion Torrent S5 sequencer (ThermoFisher, #A27212) and 500 flows were applied for each sequencing run. The second Chip was stored at 4ºC during the first sequencing run and removed to room temperature for 30 minutes prior to running the second Chip

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent S5

Data processing

Raw sequencing data from the Ion S5 was analyzed using the Ion Torrent Suite software v5.6 to generate normalized count files for each condition. Files were loaded into RStudio and count tables generated to measure differential gene expression between different conditions (e.g. RAF1 mutant vs isogenic control, ERK5 or MEK1/2 inhibitors vs vehicle) using the DESeq2 package2. Estimations of dispersion were plotted from the count data using the plotDispEsts function. Data were regularized log transformed and a sample matrix was visualized to compare different replicates and conditions. Principal components analysis (PCA) was performed using the prcomp function to separate out different conditions. Assuming a negative binomial distribution of the count data, the DESeq function was used to estimate the fold change of differentially expressed genes between different conditions, and p values were adjusted using the Benjamini Hochberg method.
Genome_build: hg38
Supplementary_files_format_and_content: Excel files representing differential gene expression between different conditions (e.g. RAF1 mutant vs isogenic control, ERK5 or MEK1/2 inhibitors vs vehicle) generating using the DESeq2 package

Submission date

May 13, 2019

Last update date

Jun 15, 2019

Contact name

Fabrice Jaffre

Organization name

Weill Cornell Medicine

Department

Surgery

Street address

1300 York Ave