|
| Status |
Public on Jan 01, 2020 |
| Title |
Chip_Input |
| Sample type |
SRA |
| |
|
| Source name |
Asynchronous-asexual stage parasites
|
| Organism |
Plasmodium falciparum 3D7 |
| Characteristics |
tissue: Whole body (trophozoite and schizont enriched)
Stage: Asexual
chip antibody: none
|
| Treatment protocol |
Asynchronous stage parasites (trophozoite and schizont enriched) with 10% parasitemia were harvested and fixed with 1% formaldehyde at 37°C for 10 minutes. To prevent the over crosslinking, excess formaldehyde was quenched by addition of glycine to a final concentration of 150 mM. After that, the parasitized RBC’s were washed twice with 1x PBS followed by saponin lysis to remove the RBCs.
|
| Growth protocol |
Parasites were cultivated in O+ human erythrocytes at 3% haematocrit in RPMI 1640 medium supplemented with 0.5% Albumax II, 0.2% sodium bicarbonate, 27.2 mg/litre hypoxanthine and 50 mg/litre Gentamycin. The parasites were grown at 37°C in an atmosphere of 5% CO2, 5% O2 and 90% nitrogen. Synchronization of parasites was carried out using 5% sorbitol treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Parasites were resuspended in ice cold SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) supplemented with PMSF (100μM/ml) and protease inhibitor cocktail for 10 minutes on ice. Parasite lysate was then sonicated to generate the sheared chromatin of ~500bp and centrifuged at 1000g for 15 minutes. After the removal of debris from the lysate, the lysate was further diluted 10 times in ChIP dilution buffer (0.01% SDS, 0.1% Triton-X 100, 1.2mM EDTA, 16.7mM Tris, pH 8.1, and 150mM NaCl). Subsequently, the parasite lysates were precleared using protein A sepharose beads at 4°C for 1 hour. Cleared chromatins were incubated with pre-immune sera and immune GCN5 sera for overnight at 4°C on rotating wheel.
The paired-end sequencing library was prepared using Illumina TruSeq Nano DNA LT Library Preparation Kit. 200ng of each DNA was fragmented by Covaris S2. Covaris shearing generates DNA fragments with 3' or 5' overhangs. The fragments were then subjected to end-repair. This process converts the overhangs resulting from fragmentation into blunt ends using End Repair Mix. The 3' to 5' Exonuclease activity of this mix removes the 3' overhangs and the 5' to 3' polymerase activity fills in the 5' overhangs. A single "A" nucleotide is added to the 3' ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single "T" nucleotide on the 3' end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation. Indexing adapters were ligated to the ends of the DNA fragments, preparing them for hybridization onto a flow cell. The ligated products were purified using Ampure XP beads. The product was PCR amplified as described in the kit protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Input
|
| Data processing |
The high quality paired end ChIP seq reads generated through hi-throughput sequencing were mapped with the reference genome (PlasmoDB-41_Pfalciparum 3D7) using Bowtie2 (v2.1.0).
PfGCN5 peak calling using MACS2 (v2.1.2) with 0.05 p-value cutoff.
Genome_build: Plasmodium_falciparum_3D7, version=2015-06-18
Supplementary_files_format_and_content: GCN5_NP.bedgraph file contains chromosome name, peaks start site, peaks end site and score value.
Supplementary_files_format_and_content: GCN5_NP_peaks.narrowPeak
|
| |
|
| Submission date |
Dec 31, 2019 |
| Last update date |
Jan 01, 2020 |
| Contact name |
Sunita Verma |
| E-mail(s) |
sunita85_sit@jnu.ac.in
|
| Phone |
+91-9205738570
|
| Organization name |
Jawaharlal Nehru Univesity
|
| Department |
School of Computational and Integrative Sciences
|
| Lab |
14
|
| Street address |
JNU Campus
|
| City |
New Delhi |
| State/province |
Delhi |
| ZIP/Postal code |
110067 |
| Country |
India |
| |
|
| Platform ID |
GPL24926 |
| Series (1) |
| GSE142803 |
Identification of genome-wide binding sites of Plasmodium falciparum GCN5-acetyltransferase |
|
| Relations |
| BioSample |
SAMN13703877 |
| SRA |
SRX7484198 |
