|
| Status |
Public on Sep 05, 2013 |
| Title |
RNA-seq in N2 mixed-stage embryos treated with NPP-13 RNAi (BC184) |
| Sample type |
SRA |
| |
|
| Source name |
Mixed-stage embryos
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
treatment: NPP-13 RNAi
isolation: RiboMinus RNA (rRNA-depleted total RNA)
|
| Treatment protocol |
For RNAi, the N2 strain worms were first arrested at the L1 larva stage in the liquid S medium for synchronization. Synchronized L1 worms were fed with a bacteria strain HB101, which do not contain induction vectors, at 20ºC for 36 hours (L4 stage) in the liquid S medium. L4 worms were harvested, washed and transferred to a new S medium supplemented with a bacteria strain HT115 expressing interfering double-strand RNA from L4440 vector under IPTG (RNAi bacteria). Worms were grown with the RNAi bacteria at 20ºC until the gravid stage. Embryos were harvested from the gravid adults by hypochlorite treatment and used in experiments. In parallel, worms were fed with HT115 bacteria bearing L4440 vector without target DNA insertion (empty vector RNAi).
|
| Growth protocol |
The C. elegans N2 strain animals in the liquid S-medium were fed with bacteria HB101 until becoming gravid adults. Mixed-stage embryos were harvested from the adults by hypochlorite treatment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
First, total RNA were treated with DNase I, and then ribosomal RNA were depleted from the sample using Invitrogen?s RiboMinus Eukaryote Kit for RNA-seq (cat# A10837-08). rRNA-depleted RNA were recovered by ethanol precipitation. RNA were then fragmented by incubating in buffer containing Zn2+ at 70 oC for 5 min (Ambion, Fragmentation Reagent cat# AM8740), followed by ethanol precipitation. Fragmented RNA were used to synthesize double-strand cDNA using the SuperScript II kit (Invitrogen, cat #18064-014) and DNA polymerase I (Enzymatics P705L). cDNA were recovered from the reaction using Qiagen?s Mini elute columns.
DNA was blunt-ended with End Repair Enzyme mix (Epicentre, Madison) and A-overhanged with Exo(-) Klenow fragment in the presence of dATP. DNA fragments were ligated with adaptors. DNA was amplified by PCR with single-end or pair-end primers. Amplicon was loaded on an agarose gel, and DNA at an indicated size range was recovered from the gel.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
seq-RNA_RiNPP13_KI2126_N2_MXEMB
Barcode: na
|
| Data processing |
Unique sequencing reads were aligned to the C. elegans reference genome (ce6, WS190) using tophat
Coverage of reads in bedgraph format was obtained using Bedtools
Processed data files are in bedGraph format representing overlappaing read counts in the genome.
Genome_build: ce6, WS190
|
| |
|
| Submission date |
Dec 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Kohta Ikegami |
| E-mail(s) |
kohta.ikegami@cchmc.org
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Department |
Division of Molecular Cardiovascular Biology
|
| Lab |
Ikegami Lab
|
| Street address |
240 Albert Sabin Way, Cincinnati, OH 45229
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL13657 |
| Series (2) |
| GSE42714 |
Integral nuclear pore proteins bind to Pol III genes and are required for Pol III transcript processing in C. elegans [seq] |
| GSE42741 |
Chromatin Immunoprecipitation of Nuclear Pore Proteins in C. elegans |
|
| Relations |
| SRA |
SRX208780 |
| BioSample |
SAMN01823263 |
