|
| Status |
Public on Sep 05, 2013 |
| Title |
IMB-1 ChIP-seq in N2 mixed-stage embryos (KI026) |
| Sample type |
SRA |
| |
|
| Source name |
Mixed-stage embryos
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
strain: N2
treatment: no treatment
chip antibody: SDQ4154_IMB1 [not commercial]
|
| 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 |
genomic DNA |
| Extraction protocol |
The C. elegans N2 strain mixed-stage embryos were cross-linked in 2% formaldehyde for 30 min at 25ºC. Chromatin extract was prepared by sonication in FA buffer (50 mM HEPES/KOH pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate; 150 mM NaCl). Five to ten micrograms of antibodies were immobilized on sepharose beads and incubated with the chromatin extract for >12 hours at 4ºC. After washing the immunoprecipitants followed by RNase treatment and reverse-crosslinking, DNA were extracted and purified.
DNA was blunt-ended 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 primers. Amplicon was loaded on an agarose gel, and DNA at 200-500 bp was recovered from the gel.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
seq-SDQ4154_IMB1_EE9_N2_MXEMB
Barcode: na
|
| Data processing |
Unique sequencing reads were aligned to the C. elegans reference genome (ce6, WS190) using bowtie, allowing up to two mismatches
ChIP-seq reads were extended to the original DNA fragment size estimated computationally as the distance between positive- and negative-strand reads with the highest cross-correlation.
The number of overlapping reads per base was computed using 'basealigncount' function in Zinba (Rashid et al., Genome Biology 2011)
Processed data files are in wig format representing overlappaing read counts at each base 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 |
GPL9269 |
| 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 |
SRX208764 |
| BioSample |
SAMN01823247 |
