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Genome-wide miRNA turnover profile after transcription inhibition in HDMYZ cells with biological triplicate

ABSTRACT: HDMYZ cells were treated with 4ug/ml ActD for 0, 1, 4 and 12 hours. Small RNAs of 15-40 bases were gel-purified from 5 ug total RNA, and subjected to multiplex Illumina small RNA library preparation. Small RNA libraries were sequenced on a HiSeq2000 (Illumina).

ORGANISM(S): Homo sapiens

PROVIDER: GSE60036 | GEO | 2015/02/05

SECONDARY ACCESSION(S): PRJNA257358

REPOSITORIES: GEO

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Genome-wide miRNA turnover profile after transcription inhibition in HDMYZ cells with biological triplicate

Project description:HDMYZ cells were treated with 4ug/ml ActD for 0, 1, 4 and 12 hours. Small RNAs of 15-40 bases were gel-purified from 5 ug total RNA, and subjected to multiplex Illumina small RNA library preparation. Small RNA libraries were sequenced on a HiSeq2000 (Illumina). Measure miRNA expression level at 0, 1, 4, 12 hours post ActinomycinD treatment to examine miRNA turnover pattern.

2015-02-05 | E-GEOD-60036 | biostudies-arrayexpress

Genome-wide miRNA turnover profile in HDMYZ cells after transcription inhibition

Project description:HDMYZ cells were treated with 2ug/ml ActD for 0, 4 and 12 hours. Small RNAs of 15-40 bases were gel-purified from 10 ug total RNA, and subjected to multiplex Illumina small RNA library preparation. Small RNA libraries were sequenced on a HiSeq2000 (Illumina) with 3 samples per lane. To quantify miRNA and isoform abundance, sequence reads were processed by the miRDeep2 package, with the following modifications. First, to remove adaptor sequence, we removed both the main adaptor sequence present in the sequencing reads, as well as the second most abundant adaptor variant. In addition, we did not restrict the size of small RNAs during adaptor removal. Second, we used miRBase v18 for mapping the reads. Third, for quantifying miRNA and isoform frequency, we limited reads to more or equal to 15 bases in length with zero mis-match during mapping. The number of reads that were mapped to known miRNAs was used to normalize read frequencies for each miRNA or each miRNA isoform. For quantification purposes, we only considered miRNAs or isoforms that had frequency >= 1x10e-6 in samples without ActD treatment, which correspond to ~21-30 reads in raw count. These miRNAs or isoforms were referred to as reliably quantifiable.To analyze mapping to the genome, we removed reads that mapped to miRNA precursors. The rest of the reads were then mapped to the genome with Bowtie.

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Genome-wide miRNA turnover profile in various cell types after transcription inhibition

Project description:We measured miRNA turnover rates in 8 mammalian cell types post transcription inhibitors ActinomycinD (ActD) or 5,6-Dichlorobenzimidazole 1-M-NM-2-D-ribofuranoside (DRB) treatment with miRNA expression profiling, generating 218 expression profiles on 528 endogenous miRNAs. Cells were plated in six-well plates at around 200,000 cells/well. A media change was performed one day after plating. ActD and DRB treatment were started after confluency (around two days after plating) for 0, 2, 4, 6, 12, and 24 hours post treatment with biological duplicate (ActD treatment) or biological triplicate (DRB treatment). The concentration of ActD and DRB for different cell lines were titrated to achieve rapid c-myc down-regulation after 2 hours of ActD and DRB treatment. After treatment at indicated time points, cells were washed with PBS once and lysed in TriZol reagent immediately (Invitrogen). RNA extraction was performed following the manufacturerM-bM-^@M-^Ys protocol. miRNA profiling was performed using a Luminex bead-based method.

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