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Protocol Dependence of Sequencing-based Gene Expression Measurements

ABSTRACT: RNA samples from human tissues (brain and liver) and cell lines (K562 and HL60) were used to assess RNA fragmentation, RNA fractionation, cDNA synthesis, and single versus multiple tag counting. Technical replicates and different cDNA synthesis protocols were compared. Though protocols employing polyA RNA selection generate the highest number of non-ribosomal reads and the most precise measurements for coding transcripts, such protocols were found to detect only a fraction of the non-ribosomal RNA in human cells. PolyA RNA excludes thousands of annotated and even more unannotated transcripts, resulting in an incomplete view of the transcriptome. Ribosomal-depleted RNA provides a more cost-effective method for generating complete transcriptome coverage. Expression measurements using single tag counting provided advantages for assessing gene expression and for detecting short RNAs relative to multi-read protocols. Detection of short RNAs was also hampered by RNA fragmentation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE28123 | GEO | 2011/03/24

SECONDARY ACCESSION(S): PRJNA139845

REPOSITORIES: GEO

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Project description:The tracks show enrichment of RNA sequence tags generated by high throughput sequencing (RNA-seq) and mapped to the human genome. Double stranded cDNA was synthesized from polyadenylated RNA (polyA+) . PCR amplified, adapter ligated cDNA, 150-300nt long, was sequenced on an Illumina GA sequencer. Where designated, cell lines received specific treatments prior to RNA isolation. As indicated, K562 cells were treated with either interferon-a or interferon-g for 30 minutes or 6 hours. These experiments were carried out in conjunction with ChIP-Seq experiments on the transcription factors STAT1 and STAT2 in order to examine the effects that inducers of a specific transcriptional response might have on gene expression and on transcription factor binding site discovery. K562 cells were treated with a-amanitin in order to examine the effects of RNA polymerase II inhibition on RNA polymerase III-mediated transcription. This track shows expression data generated as confirmation of the SYDH TFBS (http://genome-preview.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=wgEncodeSydhTfbs) tracks currently available on genome-preview. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Total RNA was extracted using TRIzol reagents (15596-018, Life Tech), following the manufacturer's protocol. For polyA+ samples, polyadenylated RNA was purified using the MicroPoly(A) Purist kit (AM1919, Life Tech) and fragmented using RNA Fragmentation Reagent (AM8740, Life Tech). Illumina adapters were ligated to double stranded cDNA which was synthesized using reagents from Life Tech (11917-010). PCR amplified adapter ligated cDNA (150-300 bp) was sequenced using Illumina GA. Sequence reads of 27-33nt long with 0-2 mismatches were mapped to the genome. The signal height corresponds to the number of overlapping fragments at each nucleotide position in the genome. Samples originally mapped to the hg18 version of the human genome were remapped to hg19 using the BWA aligner, version 0.5.7.

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Protocol Dependence of Sequencing-based Gene Expression Measurements

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