|
| Status |
Public on Jan 26, 2016 |
| Title |
Dcr2 RNAi sm RNA-seq rep2 |
| Sample type |
SRA |
| |
|
| Source name |
tissue culture cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Dmel-2
|
| Treatment protocol |
RNAi was performed as described (Sullivan, et. al., Mol. Cell, 2009)
|
| Growth protocol |
Dmel-2 cells were grown in Sf-900 serum free media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from 8 x 106 Drosophila Dmel-2 tissue culture cells was isolated using QIAzol Lysis Reagent (Qiagen). Total RNA was fractionated into large (>200 nts) and small (<200 nts) fractions using RNeasy Mini spin columns and RNeasy MinElute spin columns, respectively (Qiagen). DNA was removed from the large fraction by on-column DNase digestion (Qiagen). 28S, 18S and 5S rRNAs were depleted from 5 µg of each large RNA fraction using the Ribo-Zero Magnetic Kit (Epicentre). 2S rRNA was depleted from the small RNA fraction according to Seitz et al. (SEITZ et al. 2008) with the following modifications. 0.1 nMoles 2S rRNA complementary oligo were bound to 500 µg streptavidin beads in 1 mL 0.5x SSC for one hour at 4oC. The beads were then washed 5X in 0.5X SSC followed by 5 minute incubation at 65oC to remove secondary structure. 2 µg of the small RNA fraction were diluted to 12.5 ng/µl and 160 µl were added to the bead slurry.
RNA-seq libraries were prepared in triplicate from 35 ng of the rRNA-depleted large RNA fraction using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB). smRNA-seq libraries were prepared in triplicate from ~475 ng of the 2S rRNA-depleted small RNA fraction using the NEBNext Small RNA Library Prep Set for Illumina (NEB).
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina MiSeq |
| |
|
| Description |
rRNA depleted Small RNA
|
| Data processing |
All adapter sequences were trimmed and the libraries cleaned using Cutadapt (MARTIN 2011).We aggressively trimmed the siRNA reads to 25 nts from the 5’ end following adapter removal to filter remaining rRNAs, snoRNAs, snRNAs and tRNAs out of the data set before mapping. All data sets were mapped using the RNA-seq Unified Mapper (RUM) (GRANT et al. 2011). The NEB kit used to prepare the RNA-seq samples produces libraries with high directionality and RUM utilized this feature to strand specifically map the RNA-seq reads. RUM separated unique and non-uniquely mapping sequences into separate output files that could be further analyzed. The UCSC genome browser (www.genome.ucsc.edu) was used to visualize the non-uniquely mapping bedgraph output files.
Genome_build: dm3
Supplementary_files_format_and_content: Text files with normalized readcounts unique and non-unique RNA-seq and small RNA-seq reads.
|
| |
|
| Submission date |
Apr 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Mindy Steiniger |
| E-mail(s) |
steinigerm@umsl.edu
|
| Organization name |
University of Missouri-St. Louis
|
| Street address |
1 University Blvd.
|
| City |
St. Louis |
| ZIP/Postal code |
63121 |
| Country |
USA |
| |
|
| Platform ID |
GPL16479 |
| Series (1) |
| GSE67725 |
Antisense transcription of retro transposable elements in Drosophila: The origin of endogenous small interfering RNA precursors |
|
| Relations |
| BioSample |
SAMN03468709 |
| SRA |
SRX984891 |
