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Sample GSM1517072

Query DataSets for GSM1517072

Status

Public on Mar 01, 2016

Title

RNA-seq, shControl, without TGF-beta

Sample type

SRA

Source name

A549

Organism

Homo sapiens

Characteristics

cell line: A549
shRNA: Negative control
rip: no
tgf-beta treatment: no
histology: lung adenocarcinoma

Treatment protocol

For RNA-seq, shRNAs were introduced into cells using lentiviral vectors. Cells were stimulated with 2.5 ng/ml TGF-beta or left untreated for 24 h before harvest. For RIP-seq, A549 cells were cultured in 10-cm dish before lysis.

Growth protocol

A549 cells were maintained in Dulbecco's modified Eagle's medium (DMEM #11965; Thermo Scientific) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin G, and 100 µg/ml streptomycin. Cells were grown in a humidified atmosphere with 5% CO2 at 37°C.

Extracted molecule

polyA RNA

Extraction protocol

For RNA-seq, total RNAs were extracted as described previously (Arase et al., 2014, Cancer Sci). RIP samples were obtained from A549 cells following the manufacturer's protocol (Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore)).
For RNA-seq, Libraries were prepared using Dynabeads mRNA DIRECT Purification Kit (Life Technologies) and Ion Total RNA-seq kit v2 (Thermo Scientific). For RIP-seq, Ion Total RNA-seq kit v2 (Thermo Scientific) was used. Sequencing was performed using Ion PI Chip Kit v2, PI Template OT2 200 Kit v3 and Ion PI Sequencing 200 Kit v3 (Thermo Scientific).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent Proton

Description

control shRNA is stably expressed, without TGF-beta stimulation

Data processing

Reads were processed by Tophat2 (version 2.0.10) and Bowtie2 (version 2.1.0) using hg19 version of index files. Parameters used are as follows: -a 6 --segment-length 25 --no-coverage-search --read-realign-edit-dist 0 -G.
FPKM calculation was performed by using Cuffdiff (cufflinks version 2.1.1, with options -m 200 -u).
Genome_build: hg19
Supplementary_files_format_and_content: For RNA-seq, expression data of the four samples were summarized into single processed file by Cuffdiff and are in RNA-seq.genes.fpkm_tracking.txt. For RIP-seq, calculated FPKM values of immunoprecipitated RNA of the four samples were summarized into single processed file by Cuffdiff and are in RIP-seq.genes.fpkm_tracking.txt.

Submission date

Sep 30, 2014

Last update date

May 15, 2019

Contact name

Daizo Koinuma

E-mail(s)

d-koinuma@umin.ac.jp

Organization name

University of Tokyo

Department

Pathology