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Sample GSM1668998

Query DataSets for GSM1668998

Status

Public on Apr 28, 2017

Title

R273H-mutant-Replicate2

Sample type

SRA

Source name

Human non-small cell lung carcinoma cell line derived from the lymph node

Organism

Homo sapiens

Characteristics

cell line: H1299
cell type: non-small cell lung carcinoma
passages: 15-20
genotype/variation: mutant p53R273H expressing

Treatment protocol

Human non-small cell lung carcinoma cell line H1299 either harboring an empty vector (EV) or stably expressing R273H mutant p53 (p53R273H ) were used in this study. The cells were left untreated or treated with 10 µM Etoposide (Sigma Aldrich, St. Louis, USA) for 24 hours before harvesting.

Growth protocol

To generate mutant p53R273H expressing stable H1299 cell line, cells were first transfected with pCMV expression vector containing R273H mutant p53 cDNA (pCMV-Neo-Bam-p53 R273H, kindly provided by Dr. Bert Vogelstein, Johns Hopkins Kimmel Cancer Center, Baltimore, MD 21205, USA) using Lipofectamine2000 Reagent (Invitrogen, Thermo Fisher Scientific Inc., MA USA). Forty-eight hours post transfection, cells were sub-cultured at 1:6 and allowed to grow in media supplemented with G418 ( Life Technologies, Thermo Fisher Scientific Inc., MA USA) at a final concentration of 800 µg/ml. After 15-20 days, G418 resistant colonies were picked up and propagated in G418 containing (400 µg/ml) media to generate stable cell lines expressing mutant p53R273H . The H1299 cell line infected with an empty vector was kindly provided by Prof. Varda Rotter (Weizmann Institute of Science, Rehovot, Israel). All cell lines were cultured in RPMI 1640 medium (Life Technologies, Thermo Fisher Scientific Inc., MA USA) supplemented with 10% fetal calf serum, 1% Pen Strep and 0.006% Gentamicin (Life Technologies, Thermo Fisher Scientific Inc., MA USA). The cells were treated with 10 mM Etoposide (Sigma Aldrich, St. Louis, USA) for 24 hours before harvesting.

Extracted molecule

total RNA

Extraction protocol

Small RNA fractions from cells were isolated using PureLink miRNA Isolation kit (Ambion, USA) according to the manufacturer’s protocol. Briefly, in a two-column based purification method, cells were first resuspended in binding buffer containing guanidine isothiocyanate and mixed with ethanol (final concentration of 35%). The mix was then passed through a spin column and flow through containing small RNAs was collected. Ethanol (final concentration 70%) was added to the flow through fraction and the mix was processed through a silica based column to bind the small RNAs to the silica membrane. The impurities were subsequently removed by washing the column with wash buffer and small RNAs were eluted in RNase free water.
Total RNA-Seq kit V2.0 (Life Technologies, Thermo Fisher Scientific Inc., MA USA) was used to prepare small RNA cDNA libraries. Briefly, small RNAs (~150-250 ng) were first mixed with Hybridization Solution and Ion Adaptor Mix v2 and hybridization reaction was performed in thermal cycler. Ligation enzyme mix was then added to the hybridization reactions and incubated in a thermal cycler at 16°C for 16 hours. Adapter ligated small RNA samples were subsequently reverse transcribed with Ion RT Primer v2 and SuperScript® III Enzyme Mix to make cDNAs. The cDNA products were then purified and size-selected using the magnetic bead cleanup module and PCR amplified with Ion 5’ and 3’ PCR Primers. The amplified DNAs were quantified and assessed for size distribution using Agilent® DNA 1000 Kit in Agilent® 2100 Bioanalyzer. Each cDNA library was then clonally amplified on Ion Sphere™ Particles (ISPs) by emulsion PCR in Ion OneTouch™ System using Ion OneTouch™ 200 Template Kit v2 DL (Life Technologies, Thermo Fisher Scientific Inc., MA USA). Enrichment of the template positive ion spheres was carried out in Ion OneTouch™ Enrichment System. Template enriched ion sphere particles were subsequently sequenced on the Ion PGM™ sequencer using the Ion PGM™ 200 Sequencing Kit ((Life Technologies, Thermo Fisher Scientific Inc., MA USA) and Ion 316™ Chip.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Ion Torrent PGM

Description

mutant p53R273H expressing cell line

Data processing

Raw sequence reads were pre-processed using FASTX-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) to generate high quality reads.
Reads were trimmed at 3’ end and checked for base quality value calculated using Phred score (Q≥ 20). We only considered the sequencing reads between 17 bp to 35 bp read length.
Processed reads were then mapped to rRNAs, tRNAs, 3’ adapter sequence and mature miRNA sequences (miRBase 20) using the SHRiMP aligner (http://compbio.cs.toronto.edu/shrimp).
After the alignment, an in-house Perl script was used for counting the reads mapping to each of miRNA, tRNA, rRNA and adapter sequence.
miRNAs having 3 or more reads were used for differential expression analysis using R package DESeq.
Genome_build: hg19
Supplementary_files_format_and_content: DESeq generated csv files contain normalized read counts and differential expression of miRNAs between different pairs.
Supplementary_files_format_and_content: normalized_EVUT_vs_EVT.csv: Normalized read counts of two replicates of both EVU and EVT
Supplementary_files_format_and_content: differential_EVUT_vs_EVT.csv: Differential expression betwqeen EVU and EVT
Supplementary_files_format_and_content: normalized_EV_vs_R273H.csv: Normalized read counts of two replicates of both EVU and R273H-mutant
Supplementary_files_format_and_content: differential_EV_vs_R273H.csv: Differential expression between EVU and R273H-mutant
Supplementary_files_format_and_content: normalized_R273HUT_vs_R273HT.csv: Normalized read counts of two replicates of both R273H-mutant and R273HT-mutant
Supplementary_files_format_and_content: differential_R273HUT_vs_R273HT.csv: Differential expression between R273H-mutant and R273HT-mutant

Submission date

Apr 28, 2015

Last update date

May 15, 2019

Contact name

Raghunath Chatterjee

E-mail(s)

rchatterjee@isical.ac.in

Organization name

Indian Statistical institute

Department

Human Genetics Unit