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Sample GSM1359516

Query DataSets for GSM1359516

Status

Public on Mar 29, 2014

Title

K369R

Sample type

SRA

Source name

Xenografts of nude mice BALB/c, KLF5 mutant

Organism

Homo sapiens

Characteristics

tissue: tumor
cell type: prostate cancer cell line DU-145 xenograft
expression: KLF5 mutant K369R

Treatment protocol

After isolating, tumors were flash frozen in liquid nitrogen.

Growth protocol

Male BALB/c nude mice 3- to 4-weeks-old were used in this assay. Cancer cells were resuspended in a mixture of PBS and Matrigel (equal volumes) at 2 x 10^7 cells/ml. 100 µl of cells were injected subcutaneously into both flanks. At day 38 after injection, mice were sacrificed, and tumors were surgically isolated.

Extracted molecule

total RNA

Extraction protocol

RNA was harvested using TRIZOL reagent (Invitrogen, Beijing, China). 4ug total RNA of each sample was pooled and purified by RNeasy Micro kit (catalogue #74004, Qiagen), respectively. The quality and concentration of RNA samples were determined by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, US). 10 μg total RNA of each pool was used for library construction and RNA-Seq.
Non-directional RNA-Seq libraries (300nt average insert size) of three pools were established following the manufacturer's instruction of TruSeq RNA-Seq Library Prep Kit v2 (Illumina, SanDiego, CA, US) and quantified by Qubit 2.0 Fluorometer (Invitrogen) and validated by Agilent 2100 Bioanalyzer. Clusters were generated by cBot with the libraries diluted to 20 pM and subsequently sequenced on HiSeqTM 2500 (Illumina) platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Basecalls performed using CASAVA version 1.8.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence using FASTX-Toolkit version 0.0.13 and Perl version 5.8.8.
mRNA-seq reads were aligned to the UCSC hg19 genome assembly using Tophat version 2.0.9 with parameters: -r 100 -I 50000 --max-segment-intron 50000 -a 10 -g 1
Gene abundance measurements were generated and normalized using Cufflink version 2.1.1.Linux_x86_64; the normalized standard were Fragments Per Kilobase of exon per Megabase of library size (FPKM).
Differential expression were analysed using DEGseq function package in R program version 2.10.0.
Genome_build: GRCh37
Supplementary_files_format_and_content: Tab-delimited text file includes normalized FPKM values and raw fragment counts for each Sample.

Submission date

Mar 28, 2014

Last update date

May 15, 2019

Contact name

jintang dong

E-mail(s)

jtdong@nankai.edu.cn

Organization name

Nankai university

Street address

Weijin road