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Sample GSM802465 Query DataSets for GSM802465
Status Public on Dec 05, 2012
Title Non-tumor sample 5N
Sample type SRA
 
Source name esophageal cell type
Organism Homo sapiens
Characteristics tissue: non-tumor
Growth protocol Esophageal squamous cell carcinoma and adjacent non-tumorous tissues were obtained from patients immediately following resection, snapped-frozen in liquid nitrogen and stored in -80C freezer prior to RNA extraction.
Extracted molecule total RNA
Extraction protocol The total RNA samples were treated with DNA-free Kit (Ambion, Inc. TX, USA) to digest the DNA. Large 18S and 28S rRNAs were removed from 1ug total RNA with the RiboMinusTM Transcriptome Isolation kit (Human/Mouse) (Invitrogen, Carlsbad, CA, USA). The rRNA-depleted RNA was precipitated with Pellet Paint (Novagen Inc., Madison, Wis.), quality checked on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif.). The rRNA-depleted RNA from 1 ug of total RNA was fragmented by incubation for 5 min at 94°C in 5 x Array Fragmentation Buffer (Ambion, Inc. TX, USA). The reaction was stop by chilling the tube on ice and precipitated with Pellet Paint. Double strand cDNA was synthesized with the SuperScript Double Stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA) using random hexamers according to the manufacturer's instructions. The reaction was cleaned up on a QiaQuick PCR column (Qiagen, Valencia, CA, USA). Ds cDNA fragments were repaired with DNA Terminator End Repair Kit End (Lucigen, Middleton, WI, USA) by incubation for 30 min at 30°C and then were cleaned up on a QiaQuick PCR column. The Klenow 3' to 5' exo- (NEB, Ipswich, MA) was used to add a single 'A' base to the 3'end of blunt phosphorylated DNA fragments by incubation for 30 min at 30°C. After clean-up on a QiaQuick PCR column, Illumina PE Adapter was ligated to the end of DNA fragments with the Quick Ligation Kit (NEB, Ipswich, MA) by incubation for 15 min at room temperature. The reaction was cleaned up on a QiaQuick PCR column. 180-200 bp fragments were excised from a 2% low range agarose gel. Fragments were enriched by 10 cycles using AccuPrime Pfx DNA Polymerase (Invitrogen, Carlsbad, CA, USA). PCR product was run on Novex 8% TBE polyacrylamide gel (Invitrogen, Carlsbad, CA, USA) and stained with SYBR Gold (Invitrogen, Carlsbad, CA, USA). The gel slice was cut off and cleaned up by QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA, USA). The concentration of gel purified DNA fragments was measured by using a ND-1000 UV/Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer IIx
 
Description rRNA-depleted RNA
Data processing High-throughput sequencing reads were mapped against human genome assembly (NCBI Build GRCh37). The reads with at most 2 mismatches and a maximum of 10 mappable locations were retained for calculation of gene expression. The gene expression level is measured by RPKM (number of exon reads mapped per kilobase per million mapped reads). The gene differential expression was evaluated using the t-test and Baggerley's test.
 
Submission date Sep 27, 2011
Last update date May 15, 2019
Contact name Yunjuan Bao
Organization name University of Notre Dame
Street address 1234 Notre Dame Avenue
City Notre Dame
State/province Indiana
ZIP/Postal code 46556
Country USA
 
Platform ID GPL10999
Series (1)
GSE32424 Identification of a Novel Angiogenesis and Tumor Suppressor Gene Rab25 in Esophageal Squamous Cell Carcinoma
Relations
SRA SRX099136
BioSample SAMN00727898

Supplementary file Size Download File type/resource
GSM802465_5N_prim.bam 128.9 Mb (ftp)(http) BAM
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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