Comment[ArrayExpressAccession] E-GEOD-44802 MAGE-TAB Version 1.1 Public Release Date 2013-10-01 Investigation Title Next-Generation Sequencing for Small RNA Application in Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia Comment[Submitted Name] Next-Generation Sequencing for Small RNA Application in Scrambled Control and EGFR Knockdown Cells Cultured under Normoxia and Hypoxia Experiment Description Purpose: The goal of this study is to identify the miRNA clusters that are regulated by EGFR under normoxia or hypoxia. Method: Total RNAs were extracted from HeLa cells expressing scrambled control or EGFR shRNA-E1 that cultured under normoxia or hypoxia (1% O2) for 24h. Customized Next-Generation RNA deep sequencing, including both small RNA application and whole transcriptome analysis, was performed according to the standard procedure instructed by Applied Biosystems. For small RNA analysis, library inserts were size selected between 18 and 40nts and analyzed using CLC Genomics Workbench 4.7.1. 35nt colorspace reads were trimmed of adaptor sequence and mapped against human pre-miR sequences (miRBase version 16.0). Values of reads per million mapped reads (RPM) were based on mapped reads with no more than 2 mismatches total. A read was considered to come from a mature miRNA if it mapped to pre-miRNA sequences with no more than three upstream or downstream bases, and missing no more than two upstream or downstream bases from predicted mature or mature* sequences as defined in miRBase version 16.0. All the other pre-miRNA mapped reads were assigned as pre-miRNA signal. qRT–PCR validation was performed using TaqMan and SYBR Green assays. Results: Deep sequencing analysis identified specific miRNA clusters that their maturation (miRNA processing efficacy was reflected by the relative expression of precursor miRNAs affected by EGFR compared with the relative expression of mature miRNAs affected by EGFR) were regulated by EGFR under normixa or specifically in response to hypoxia. Top miRNA candidates that regulated by EGFR in response to hypoxia were further verified by TaqMan and SYBR Green qRT-PCR assays. Conclusion: Next-Generation Deep Sequencing for small RNA analysis revealed a novel function of EGFR involved in miRNA maturation in response to hypoxic stress. RNA profiles of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) that cultured under normoxia or hypoxia (1% O2) for 24h were generated by AB SOLiD next-generation sequencing. S: HeLa expressing scrambled control cultured under normoxia; A1: HeLa expressing EGFR shRNA-E1 cultured under normoxia; HS: HeLa expressing scrambled control cultured under hypoxia for 24h; HA1: HeLa expressing EGFR shRNA-E1 cultured under hypoxia for 24h. In total, 4 biological samples with no replicates resulted in 4 small RNA profiles. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Hung Shen James Liu Liu Hung Person First Name Mien-Chie Jia Brian Xiuping Chang-Gong Mien-Chie Person Mid Initials P Person Email mhung@mdanderson.org Person Affiliation MD Anderson Cancer Center Person Address MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX, USA Person Roles submitter Protocol Name P-GSE44802-4 P-GSE44802-1 P-GSE44802-3 P-GSE44802-2 Protocol Description 50 nt colorspace reads were trimmed to 35nt. Resulting 35nt reads were trimmed of adaptor sequence and mapped against human pre-miR sequences (miRBase version 16.0). Reads per million mapped reads (RPM) values were based on mapped reads with no more than 2 mismatches total. A read was considered to come from a mature miRNA if it mapped to pre-miRNA sequences with no more than three upstream or downstream bases, and missing no more than two upstream or downstream bases from predicted mature or mature* sequences as defined in miRBase version 16.0. All the other pre-miRNA mapped reads were assigned as pre-miRNA signal. Genome_build: hg19 Supplementary_files_format_and_content: tab-delimited text files include RPM values for each miR Same density of HeLa cells expressing scrambled control (S) or EGFR shRNA-E1 (A1) were seed and allowed for 6-8h attachment. After that, half of the experimental groups (HS and HA1) were moved into hypoxia chamber (1% O2) while the other groups (S and A1) were continuingly cultured under normoxia for another 24h. Total RNA was extracted from HeLa stable clones expressing scrambled control or EGFR shRNA-E1 that were seeded at same density and cultured under normoxia or hypoxia (1% O2) for 24h using miRNeasy Mini Kit (Qiagen). All RNA samples passed quality test with RIN-values greater than 8 as measured by Agilent Technologies Bioanalyzer, were subjected to RNA deep sequencing. RNA libraries were prepared according to the standard procedures instructed by Applied Biosystems for Deep Sequencing Cells were cultured in DMEM/F12 medium with 10%FBS Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name SHRNA EXPRESSION CULTURE CONDITION Experimental Factor Type shRNA expression culture condition Comment[SecondaryAccession] GSE44802 Comment[GEOReleaseDate] 2013-10-01 Comment[ArrayExpressSubmissionDate] 2013-03-01 Comment[GEOLastUpdateDate] 2013-10-02 Comment[AEExperimentType] RNA-seq of non coding RNA Comment[SecondaryAccession] SRP019230 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR771374-SRR771377 SDRF File E-GEOD-44802.sdrf.txt