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Sample GSM3400936 Query DataSets for GSM3400936
Status Public on Feb 08, 2019
Title AP2-G schizonts input, rep 2
Sample type SRA
 
Source name AP2-G-DD strain
Organism Plasmodium falciparum
Characteristics Stage: Schizonts
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 schizont 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) [email protected]
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 SAMN10127765
SRA SRX4740123

Supplementary file Size Download File type/resource
GSM3400936_AP2-G_S_In_rep2.bigwig 11.9 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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