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Sample GSM1126809

Query DataSets for GSM1126809

Status

Public on Dec 31, 2013

Title

4sU_RNA_repeat1

Sample type

SRA

Source name

HelaS3 cells

Organism

Homo sapiens

Characteristics

cell type: tumor cells
library: 4sU RNA

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).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

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

Submission date

Apr 19, 2013

Last update date

May 15, 2019

Contact name

Shenglin Huang

Organization name

Fudan University

Street address

270 Dongan

City

Shanghai