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Sample GSM1048423 Query DataSets for GSM1048423
Status Public on Sep 05, 2013
Title TBP-1 ChIP-seq in N2 mixed-stage embryos treated with NPP-13 RNAi (BC258)
Sample type SRA
 
Source name Mixed-stage embryos
Organism Caenorhabditis elegans
Characteristics strain: N2
treatment: NPP-13 RNAi
chip antibody: SDQ0839_TBP1 [Novus Biologicals, catalog# 29610002, lot# G3048-022, Animal SDQ0839]
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 HiSeq 2000
 
Description seq-SDQ0839_TBP1_RiNPP13_KI2183_N2_MXEMB
Barcode: CGAGAAG
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 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 SRX208777
BioSample SAMN01823260

Supplementary file Size Download File type/resource
GSM1048423_BC258_TBP1_RiNPP13_ce6_BTm1x240_COUNT.wig.gz 15.7 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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