Comment[ArrayExpressAccession] E-GEOD-56648 MAGE-TAB Version 1.1 Public Release Date 2014-04-10 Investigation Title Human nuclear Dicer restricts the deleterious accumulation of endogenous double strand RNA Comment[Submitted Name] Human nuclear Dicer restricts the deleterious accumulation of endogenous double strand RNA Experiment Description Dicer is a central enzymatic player in RNA interference (RNAi) pathways that acts to regulate gene expression in nearly all eukaryotes. Although the cytoplasmic function of Dicer is well documented in mammals, there is little known about any possible nuclear function. Here we show that Dicer is present in both the nucleus and cytoplasm, but that its nuclear levels are tightly regulated. In its nuclear manifestation Dicer interacts with RNA polymerase II (Pol II) at actively-transcribed gene loci. Loss of Dicer causes the appearance of endogenous dsRNA, leading to induction of the interferon response pathway and consequent cell death. Our results suggest that Pol II-associated Dicer restricts endogenous dsRNA formation from overlapping non-coding RNA transcription units. Failure to do so has catastrophic effects on cell function. Taken together, we present here a micro (mi)RNA independent role for human nuclear Dicer. DICER ChIP-seq profile in wt 293 HEK cells, and dsRNA-seq profile in wt and DICER-depleted 293 HEK cells Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Kamieniarz-Gdula White Schlackow Kamieniarz-Gdula Proudfoot Gullerova Person First Name Kinga Eleanor Margarita Kinga Nicholas Monika Person Email geo@ncbi.nlm.nih.gov Person Affiliation University of Oxford Person Address Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, United Kingdom Person Roles submitter Protocol Name P-GSE56648-2 P-GSE56648-4 P-GSE56648-1 P-GSE56648-3 Protocol Description The stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8). After passing Solexa CHASTITY quality filter, the clean reads were aligned to human genome (UCSC HG19) using BOWTIE software (V0.12.7). The uniquely mapped reads were used for peak detection by MACS v1.4 (Model-based Analysis of ChIP-Seq) software. Statistically significant ChIP-enriched regions (peaks) were identified by comparison to a Poisson background model (Cut-off p-value=10-5). MACS called peaks were filtered, and only those with score > 100 were retained for further analysis (file Dicer_peaks.bed) genome build hg19 supplementary files format and content: IP_HG19_bowtie_alignment.sam.gz aligned reads SAM format; Dicer_peaks.bed MACS-called and filtered peaks. Basecalling was by Illumina's bcl2fastq v1.8.4, with internal chastity filter. Parameters for configureBclToFastq:.pl --ignore-missing-bcl --fastq-cluster-count 0 --no-eamss --use-bases-mask Y*,I8,Y* --mismatches 1 --ignore-missing-stats --ignore-missing-control Mapping was by BWA v0.7 followed by stampy v1.0.21. Parameters for bwa: -t 8 -q10 Parameters for stampy: defaults, except: -t 8 --bamkeepgoodreads Processed bam file of WTCHG_64954_282 and WTCHG_64954_283 to wiggle type format. Multiple peaks from set 282 were combined into one, if they overlayed one peak in 283 and vice versa. This was done iteratively until the peaks remained constant. Top peaks refers to the peaks where the maximum coverage is >1500 in 282 and 283 data simultaneously. genome build hg19 supplementary files format and content: peak coordinates from overlay step and top peaks Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. 10 ng of the DNA sample was used for Illumina/Solexa sequencing in the following steps: 1) DNA samples were blunt-ended with T4 DNA polymerase and Klenow polymerase; 2) a dA base was added to the 3' end of each strand by Klenow (exo minus) polymerase; 3) Illumina's genomic adapters were ligated to the DNA fragments; 4) PCR amplification was performed to enrich ligated fragments; 5) Enriched product of ~300-400bp was cut out from gel and purified with QIAquick Gel Extraction Kit. The completed libraries were quantified by Agilent 2100 Bioanalyzer. The libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cell, amplified in situ. The libraries were then sequenced on the Genome Analyzer IIx following the SBS Sequencing 36 Cycle Kit v5 protocol. total RNA was prepared using Trizol reagent, digested using T1 nuclease and resulting dsRNA was sequenced 100ng of sample was phosphatase and PNK treated, as in the Illumina mRNA Directional protocol. All clean ups (post-PNK, post-PCR) were done using Ampure XP beads. Following steps were carried out using the NEBNext Small RNA Library Prep Kit but with custom indexes designed by the WTCHG facility (Reference: Improved workflows for high throughput library preparation using the transposome-based nextera system Sarah Lamble, Elizabeth Batty, Moustafa Attar, David Buck, Rory Bowden, Gerton Lunter, Derrick Crook, Bassam El-Fahmawi, Paolo Piazza BMC Biotechnology 2013, 13:104, 20 November 2013). The concentration of each library was determined by realtime using Agilent qPCR Library and a MX3005P instrument (Agilent). Sequencing was performed on an Illumina HiSeq2000, using 50bp paired end reads. Protocol Type normalization data transformation protocol normalization data transformation protocol nucleic acid library construction protocol nucleic acid library construction protocol Experimental Factor Name DICER STATUS Experimental Factor Type dicer status Publication Title Human nuclear Dicer restricts the deleterious accumulation of endogenous double-stranded RNA. Publication Author List White E, Schlackow M, Kamieniarz-Gdula K, Proudfoot NJ, Gullerova M PubMed ID 24814348 Publication DOI 10.1038/nsmb.2827 Comment[SecondaryAccession] GSE56648 Comment[GEOReleaseDate] 2014-04-10 Comment[ArrayExpressSubmissionDate] 2014-04-09 Comment[GEOLastUpdateDate] 2014-05-27 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AEExperimentType] ChIP-seq Comment[SecondaryAccession] SRP041057 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1230840-SRR1230842 SDRF File E-GEOD-56648.sdrf.txt