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Sample GSM2817893

Query DataSets for GSM2817893

Status

Public on Sep 18, 2019

Title

HCT116 cells 120 mins post stimulation with TGF-alpha - Replicate 2

Sample type

SRA

Source name

HCT116

Organism

Homo sapiens

Characteristics

tissue: Colon
cell line: HCT116 colorectal cancer cells
treatment: 120 mins post stimulation with TGF-alpha

Treatment protocol

For the stimulation studies, cells were seeded onto 6 well plates until confluent. Cells were then starved for 18 hours and subsequently stimulated with transforming growth factor alpha (TGF-α) (0.01 µg/mL, Abcam).

Growth protocol

All cell lines were maintained in DMEM (Dulbecco’s modified Eagle’s medium, Gibco) supplemented with 10% (v/v) foetal calf serum (Assay matrix), 2mM L-glutamine (Gibco) and incubated in a humidified atmosphere of 5% CO2 at 37ᵒC. Cells were routinely split every 2-3 days.

Extracted molecule

polyA RNA

Extraction protocol

RNA was isolated from the cell lines at 0, 15, 30, 60, 90 and 120 mins post-stimulation using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA was extracted from 3 biological replicates at each timepoint. An ethanol precipitation was used to further purify the RNA. Briefly, 0.1x final volume of 3 M sodium acetate, 20 µg glycogen (Life Technologies) and 2.5X 100% ethanol was used to resuspend RNA. RNA was precipitated at -80ᵒC for a minimum of 2 hours. The precipitant was spun at 13,000 x g for 30 min at 4 ᵒC. The supernatant was removed and the pellet was washed with twice with 75% ethanol, before resuspending in a final volume of 50 µL of RNase-free water. All RNA samples were treated with DNase (Ambion) to remove any contaminating DNA from the purified RNA. Briefly, 2 Units/µL of rDNase I enzyme was added to RNA in 10x DNase I Buffer and incubated at 37ᵒC for 30 min. The reaction was inactivated by addition of DNase Inactivation Reagent and purified DNA-free RNA was extracted from the resultant supernatant. RNA concentration was determined by spectrophotometry on the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and by Qubit assay (Thermo Fisher Scientific). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent).
Total RNA was converted to strand-specific Illumina compatible sequencing libraries using the NEXTflex Rapid Directional mRNA-Seq library Kit from BIOO Scientific (Austin, Texas) as per the manufacturer’s instructions (v14.10), by staff at the SAHMRI David R. Gunn Genomics facility. Briefly, 200ng of total RNA was polyA selected and the mRNA chemically fragmented prior to reverse transcription and second strand cDNA synthesis using dUTP. The resultant cDNA was poly adenylated before the ligation of Illumina-compatible barcoded sequencing adapters. The cDNA libraries were treated with UDG to degrade the second strand and PCR amplified for 15 cycles prior to assessment using a TapeStation 2200 (Agilent) for quality and Qubit fluorescence assay for quantification. In total, 36 cDNA libraries (2 cell lines X 6 time-points X 3 replicates per timepoint) were generated for sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

HCT116_T120_Rep2

Data processing

Libraries were sequenced on the Illumina HiSeq 2500 machine using a v2 High Output 100 cycle Kit (1x 100 bp SR). The quality and number of reads for each sample were assessed with FastQC v0.11.3. Adaptors were trimmed from reads, and low quality bases with Phred scores < 28 were trimmed from ends of reads, using Trimgalore v0.4.0. Trimmed reads of less than 20 nucleotides were discarded.
Reads passing all quality control steps were aligned to the hg38 assembly of the human genome using TopHat v2.1.0 (Kim et al., 2013),allowing for up to two mismatches. Reads not uniquely aligned to the genome were discarded. HTSeq-count v0.6.0 (Anders et al., 2015) was used in the union model to assign uniquely aligned reads to Ensembl Hg38.86-annotated genes.
Data were normalized across libraries by the trimmed mean of M-values (TMM) normalization method, implemented in the R v3.2.1., Bioconductor package, EdgeR v3.10.2 (Robinson et al., 2010). Only genes that had at least 5 reads per million in at least one cell-line were analysed for evidence of differential gene expression. Differentially expressed genes were identified using the glm model implemented in EdgeR (FDR < 0.05).
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited text files include read counts for each sample

Submission date

Oct 17, 2017

Last update date

Sep 18, 2019

Contact name

Sriganesh Srihari

E-mail(s)

sriganesh.srihari@sahmri.com

Phone

+61881284066

Organization name

South Australian Health and Medical Research Institute