|
| Status |
Public on Feb 08, 2019 |
| Title |
AP2-G gametocytes input, rep 2 |
| Sample type |
SRA |
| |
|
| Source name |
AP2-G-DD strain
|
| Organism |
Plasmodium falciparum |
| Characteristics |
Stage: Stage I gametocytes
strain: AP2-G-DD
antibody: None
|
| Treatment protocol |
0.5 μM Shld1 was added at 24 hpi (12 hours prior to the schizont timepoint) to stabilise the AP2-G-DD protein.
|
| Growth protocol |
Parasites were cultured at 37°C in the presence of 5% oxygen and 7% carbon dioxide, in RPMI 1640 media supplemented with hypoxanthine, 0.5% Albumax II (Invitrogen), and 2.5 nM WR99210
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as previously described (PMID:28618269) with minor modifications. Shld1 was added to AP2-G-DD cultures at 24 hpi to stabilize AP2-G and nuclei were harvested 12 (committed schizonts), 36 (sexual rings), or 60 (stage I gametocytes) hours later. Crosslinked nuclei were then resuspended in Covaris shearing buffer (0.1% SDS, 10 mM Tris pH 8.1, 1 mM EDTA) and sonicated using an M220 ultrasonicator (Covaris) and the following conditions: 5% duty cycle, 75 W peak incident power, 200 cycles per burst, treatment time 300 seconds. The sonicated chromatin was diluted ten-fold with dilution buffer and precleared with Protein A/G magnetic beads (Pierce) for two hours. The pre-cleared material was then incubated with antibody and magnetic beads overnight, with a small volume of non-immunoprecipitated input material kept separately. For immunoprecipitations with AP2-G-DD, rat α HA (Roche 3F10) was used. The next day, beads were washed and the bound DNA eluted in elution buffer. Crosslinking was reversed by incubating overnight at 45°C and DNA was subsequently treated with RNase A at 37° for 30 minutes and Proteinase K at 45° for two hours. DNA was purified the Qiagen MinElute kit and quantified using the Qubit HS DNA assay.
Barcoded libraries for Illumina TruSeq single-end sequencing were constructed using NEBNext DNA library Prep reagents (New England Biolabs) by following the standard Illumina library preparation protocol. The DNA was first end-repaired for 30 min at 20°C, purified using Agencourt AMPure XP beads (Beckman Coulter) and then dA-tailed for 30 min at 37°C, and purified using Ampure XP beads. NEXTflex Illumina DNA barcodes (diluted 1/10) were then ligated to the DNA fragments using T4 DNA ligase at 20°C for 15 min. The resulting DNA was then size selected for 250 bp inserts using Ampure XP beads PCR-amplified (12 cycles) using Kapa HiFi (Kapa biosystems) and purified using AMPure XP beads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
AP2-G-DD gametocyte input, rep 2
|
| Data processing |
Read were trimmed using Trimmomatic 0.32.3.
Reads were mapped to the P. falciparum 3D7 genome (Pf 3D7 v28, obtained from PlasmoDB) using BWA-MEM 0.4.1.
Duplicate reads were removed using samtools 1.1.2.
MACS2 (version 2.1.1.20160309) was used to call peaks with a q value cutoff of 0.01.
Genome_build: Pf 3D7 v28
Supplementary_files_format_and_content: Bam files were converted to bigwig files using bamCoverage from the deeptools suite 3.1.2.0.0.
|
| |
|
| Submission date |
Sep 25, 2018 |
| Last update date |
Feb 10, 2019 |
| Contact name |
Manuel Llinas |
| E-mail(s) |
manuel@psu.edu
|
| Organization name |
Pennsylvania State University
|
| Department |
Biochemistry and Molecular Biology
|
| Street address |
Millennium Science Complex
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL21078 |
| Series (2) |
| GSE120448 |
PfAP2-G ChIP-seq in committed schizonts, sexual rings, and stage I gametocytes |
| GSE125566 |
Regulation of sexual differentiation is linked to invasion in malaria parasites |
|
| Relations |
| BioSample |
SAMN10127757 |
| SRA |
SRX4740131 |
