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Sample GSM1635585 Query DataSets for GSM1635585
Status Public on Mar 17, 2016
Title LV-shGAS5-mRNA
Sample type SRA
 
Source name human embryonic stem cells
Organism Homo sapiens
Characteristics cell line: H1
cell type: hESC cell line
passage: P68-75
genotype/variation: stable knockdown of GAS5 by short hairpin RNA
Treatment protocol To generate stable colonies, lentiviruses were initially added to the hESCs on the second day of passage at multiplicity of infection (MOI) 10, and continued for three days. Puromycin was added on the fifth day at the concentration of 100ng/ml for selection, and hESCs colonies that survived were passaged and analyzed by either qRT-PCR or fluorescence microscopy.
Growth protocol All hESCs were maintained in feeder-free environment using E8 medium.
Extracted molecule total RNA
Extraction protocol Total RNAs were harvested using Trizol reagent and small RNAs were harvested using miRcute miRNA extraction reagent (Tian Gen, China).
RNA-seq: The complementary DNA (cDNA) libraries for single-end sequencing were prepared using Ion Total RNA-Seq Kit v2.0 (Life Technologies) according to the manufacturer’s instructions. The cDNA libraries were then processed for the Proton Sequencing process according to the commercially available protocols. Samples were diluted and mixed, the mixture was processed on a OneTouch 2 instrument (Life Technologies) and enriched on a OneTouch 2 ES station (Life Technologies) for preparing the template-positive Ion PI™ Ion Sphere™ Particles (Life Technologies) according to Ion PI™ Template OT2 200 Kit v2.0 (Life Technologies). After enrichment, the mixed template-positive Ion PI™ Ion Sphere™ Particles of samples was loaded on to 1 P1v2 Proton Chip (Life Technologies) and sequenced on Proton Sequencers according to Ion PI Sequencing 200 Kit v2.0 (Life Technologies).
miRNA-seq: The complementary DNA (cDNA) libraries for single-end sequencing were prepared using TruseqTM Small RNA sample prep Kit (Illumina) according to the manufacturer’s instructions. 1ug small RNA was used to add 3’ adaptor and 5’ adaptor to small RNA and RT-PCR was applied to the small RNA with adaptor to achieve the 1st cDNA; Truseq (Illumina) primer was used for PCR enrichment and introducing index sequence. Size selection was applied to obtain the 145-160bp library sequence. TBS380(Picogreen) was introduced to quantify the library concentration. We applied the cluster formation on flow cell utilizing cBot and sequencing on Hiseq2000 Platform.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Ion Torrent Proton
 
Description processed data file: mRNA_All_Counts.xls
Data processing RNA-Seq-Ion Proton: Mapping of single-end reads. Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent ofbases with qualities of <13.
The clean reads were then aligned to hg19 genome using the MapSplice program (v2.1.6). In alignment, preliminary experiments were performed to optimize the alignment parameters (-s 22 -p 15 --ins 6 --del 6 --non-canonical) to provide the largest information on the AS events.
miRNA-Seq: We applied the reads filtering towards the raw reads after sequencing to achieve the clean data following the criteria: a) 30% base quality <20 b) read length < 17bp c) adaptor sequence.
utilizing the BWA software, we mapped the clean data to human miRNA database and human genome.
RNA-Seq-Hiseq: Mapping of pair-end reads. Before read mapping, clean reads were obtained from the raw reads by removing the adaptor sequences, reads with >5% ambiguous bases (noted as N) and low-quality reads containing more than 20 percent ofbases with qualities of <20.
The clean reads were then aligned to hg19 genome using the Tophat program (v2.0.11) under following parameter (-a 10 -m 0 -i 31 -I 500000 –G).
Genome_build: hg19
Supplementary_files_format_and_content:
mRNA_All_Counts.xls: processed mRNA counts data for sample LV-GAS5-mRNA, LV-NC-mRNA and LV-shGAS5-mRNA
miRNA_All_Counts.xls: processed miRNA counts data for sample LV-GAS5-miRNA and LV-NC-miRNA
LncRNA_All_RPKM.xlsx: processed lncRNA RPKMs data for sample H1-P67-lncRNA and X01-P58-lncRNA
 
Submission date Mar 17, 2015
Last update date May 15, 2019
Contact name Chen Xu
E-mail(s) chenxu8836@hotmail.com
Phone +86-13774294166
Organization name The Second Military Medical University
Street address No, 800, Rd. Xiangyin
City Shanghai
ZIP/Postal code 200433
Country China
 
Platform ID GPL17303
Series (1)
GSE66993 GAS5 controls Nodal autocrine in Human Embryonic Stem Cells Self-Renewal through competing endogenous mechanism
Relations
BioSample SAMN03421232
SRA SRX957912

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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