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SRX2426681: GSM2430236: AP2-I_Input, Rep 2; Plasmodium falciparum; ChIP-Seq
1 ILLUMINA (Illumina HiSeq 2500) run: 18.6M spots, 2.8G bases, 1.3Gb downloads

Submitted by: NCBI (GEO)
Study: PfAP2-I-GFP anti-GFP and anti-IgG ChIP-seq
show Abstracthide Abstract
To determine the genome-wide occupancy of the Plasmodium falciparum transcriptional regulator of invasion PfAP2-I (PfDd2_100013100/PF3D7_1007700), we used chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Synchronized, schizont stage, 40 hours post-invasion, cultures of parasites expressing the AP2-I-GFP fusion protein were treated with formaldehyde to crosslink proteins to DNA and harvested. After shearing the DNA, the chromatin was incubated with anti-GFP antibody or IgG (as control) for immunoprecipitation. This material was used to generate Illumina sequencing libraries. The final libraries were multiplexed with fourteen barcoded samples per lane on an Illumina HiSeq 2500 system to generate 150 base pair single-end reads. Overall design: ChIP-seq libraries were prepared using three independently collected chromatin immunoprecipitated DNA samples from Plasmodium falciparum Dd2 parasites expressing AP2-I-GFP. ChIP was performed using a polyclonal anti-GFP antibody or, as a control, the same amount of anti-IgG, resulting in 3 total samples for each chromatin sample: 1 AP2-I-GFP anti-GFP ChIPed DNA, 1 AP2-I-GFP anti-IgG ChIPed DNA, and 1 sample of input DNA. Barcoded libraries for Illumina TruSeq single-end sequencing were then prepared for each ChIPed or input DNA.
Sample: AP2-I_Input, Rep 2
SAMN06146119 • SRS1862626 • All experiments • All runs
Library:
Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: The ChIP assay was performed as described in (Lopez-Rubio, 2012) with some modifications. Briefly, synchronized schizont stage parasite cultures were saponin-lysed until complete red blood cell lysis and formaldehyde-crosslinked. The isolated chromatin was sheared in SDS lysis buffer to obtain a fragment size of 100-150bp using an M220 focused-ultrasonicator (Covaris Inc.) and the following settings: 20% duty factor, 200 cycles per burst and total treatment time of 350s. After pre-clearing the chromatin with Magna ChIP protein A+G magnetic beads (Millipore), an input sample was collected and the rest of the chromatin was incubated overnight at 4C with 1μg of polyclonal anti-GFP antibody or, as control, the same amount of IgG. The immunoprecipitated chromatin was collected with Magna ChIP protein A+G magnetic beads (Millipore), extensively washed and eluted with elution buffer. The input and ChIP samples were reverse cross-linked overnight at 45C in the presence of 0.4M NaCl and purified. ChIP-qPCR was performed as described for ChIP-seq with few exceptions (details in supplemental experimental procedures of publication). Barcoded libraries for Illumina TruSeq single-end sequencing were constructed using NEBNext DNA library preparation reagents (NEB) by following the standard Illumina library preparation protocol. The libraries were PCR-amplified using Kappa HiFi (Kappa Biosystems) and purified using Agencourt AMPure XP beads (Beckman Coulter). The quality and percentage of adaptor-ligated material of the final sequencing libraries were determined by running them on an Agilent 2100 Bioanalyzer (Agilent Technologies). The concentration of each library was determined using a Quant-iT dsDNA Broad-Range Assay kit (Invitrogen).
Experiment attributes:
GEO Accession: GSM2430236
Links:
Runs: 1 run, 18.6M spots, 2.8G bases, 1.3Gb
Run# of Spots# of BasesSizePublished
SRR511467018,599,8942.8G1.3Gb2017-06-05

ID:
3524842

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