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Status |
Public on Dec 31, 2013 |
Title |
4sU_RNA_repeat2 |
Sample type |
SRA |
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Source name |
HelaS3 cells
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Organism |
Homo sapiens |
Characteristics |
cell type: tumor cells library: 4sU RNA
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted using Trizol Reagent (Invitrogen) following the manufacturer’s instructions. Nuclear and cytoplasmic RNA were separated and extracted using the PARIS™ Kit (Ambion). Poly(A) RNA was purified with two rounds of hybridization to Dynal oligo(dT) beads (Invitrogen). Nuclear and cytoplasmic RNA samples were treated with RiboMinus™ Eukaryote Kit (Invitrogen) to remove ribosomal RNA before constructing RNA-seq libraries.The 4sU-labeled RNA was isolated by using organomercurial affinity matrix. Strand-specific RNA-seq libraries were constructed from various RNA samples. Specifically, the 4sU-labeled RNA, poly(A) RNA, nuclear RNA (ribosome minus), and cytoplasmic RNA (ribosome minus) were used for library construction. Briefly, RNA samples (about 200 ng) were fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. For second-strand cDNA synthesis, dUTP mix (without dTTP) was used which allows for the removal of the second strand. The cDNA fragments were treated with End-It DNA End Repair Kit (Epicentre) to repair the ends, then modified with Klenow fragment 3’ to 5’ exo- (NEB) to add an “A” base at the 3’end of the DNA fragments, and finally ligated to adapters with a “T” overhang. Ligated cDNA products were size selected (300-600 bp) and treated with uracil DNA glycosylase (NEB) to remove the second-strand cDNA. Purified first-strand cDNA was subjected to 15 cycles of PCR amplification. Library quality was determined on Bioanalyzer 2100 (Agilent). Finally sequencing was performed on Illumina HiSeq 2000 (2 × 100 bp paired-end).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
Illumina Casava1.7 software used for basecalling. Reads were mapped to human reference genome GRCh37/hg19 by using TopHat 1.3.1 The expression levels of mRNAs and geRNAs were calculated with FPKM (Fragments Per Kilobase per Million fragments mapped) by using Cufflink 2.0.2 Genome_build: hg19 Supplementary_files_format_and_content: bigwig files
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Submission date |
Apr 19, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Shenglin Huang |
Organization name |
Fudan University
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Street address |
270 Dongan
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City |
Shanghai |
ZIP/Postal code |
200032 |
Country |
China |
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Platform ID |
GPL11154 |
Series (1) |
GSE46228 |
Widespread Transcription beyond mRNA 3’ Ends Yields Abundant Regulatory RNAs |
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Relations |
BioSample |
SAMN02053912 |
SRA |
SRX269415 |
Supplementary data files not provided |
SRA Run Selector |
Processed data are available on Series record |
Raw data are available in SRA |
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