|
| Status |
Public on Jul 24, 2021 |
| Title |
PfCDP_ChIP_Replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Plasmodium falciparum culture
|
| Organism |
Plasmodium falciparum NF54 |
| Characteristics |
strain: NF54
chip antibody: PfCD-antibody, raised in mouse
treatment: Untreated
|
| Growth protocol |
Asexual blood stages of P. falciparum 3D7 strain was cultured at 5% hematocrit using advanced RPMI 1640 supplemented with 2 mM L- glutamine, 0.6% Albumax II and 10% human O+ Serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The parasites were released from infected RBCs by lysis with 0.15 % saponin. Crosslinked chromatin was prepared by adding 1% formaldehyde to the culture for 5 min followed by the addition of glycine to 0.125 M final concentration for 5 minutes to stop the cross-linking. Chromatin was sheared by sonication in a Bioruptor UCD-200 (Diagenode) for 10 min at 30 s intervals, power setting high, to a size of 300–800 bp. ChIP was performed by adding 1.8 µg of H3K64me3 antibodies to ChIP buffer (100 mM NaCl, 20 mM Tris pH-7.5, 6 mM EDTA, 1% Triton X- 100). The three respective RBC stage samples of wild-type 3D7 were added to the mixture and incubated at 4 °C for overnight, followed by the addition of 15 μl protein A Dynabeads and further incubated for 4 h. After washing with buffers containing 180 mM NaCl, immuno-precipitated DNA was eluted and purified using PCR purification columns.
Libraries for ChIP sequencing were prepared using NEB Next Ultra II DNA Library preparation kit. In brief, ChIP DNA and the Input DNA are subjected to various enzymatic steps for repair the ends and tailing with dA-tail followed by ligation of adapter sequences. These adapter ligated fragments are then size selected using SPRI beads. Next, the size selected fragments are indexed during limited cycle PCR to generate final libraries for paired-end sequencing. The resulting libraries are quantified before getting sequenced on Illumina HiSeq 2500/4000 system to generate 2X50 bp sequence reads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
FastQC for quality analysis and Cutadapt for adaptor trimming
ChIP seq reads were aligned to PlasmoDB-37 using Bowtie2-build
Peak calling was performed using MACS3 tool
ChIP-seq signals were background subtracted using MACS3 bdgcmp tool
multiBigwigSummary
Genome_build: PlasmoDB-37
Supplementary_files_format_and_content: Deeptools was used for viualization
Supplementary_files_format_and_content: Bamcoverage tool was used to generate bigwig files from the normalized ChIP datasets
|
| |
|
| Submission date |
Jul 22, 2021 |
| Last update date |
Jul 25, 2021 |
| Contact name |
Arumugam Rajavelu |
| E-mail(s) |
arumugam.rajavelu@iitm.ac.in
|
| Organization name |
IITM
|
| Street address |
Adayar
|
| City |
Chennai |
| State/province |
Tamil Nadu |
| ZIP/Postal code |
600036 |
| Country |
India |
| |
|
| Platform ID |
GPL30427 |
| Series (1) |
| GSE180668 |
Global mapping of PfCDP occupany on the genome of P. falciparum |
|
| Relations |
| BioSample |
SAMN20351272 |
| SRA |
SRX11523884 |
