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Sample GSM2743124

Query DataSets for GSM2743124

Status

Public on Jan 16, 2018

Title

P.vivax-HP1

Sample type

SRA

Source name

intraerythrocytic stage, schizont stage

Organism

Plasmodium vivax

Characteristics

strain: Pool of eight clinical P. vivax isolates
antibody: alpha-PvHP1

Extracted molecule

genomic DNA

Extraction protocol

Chromatin immunoprecipitations: For each ChIP reaction, sonicated chromatin containing 500 ng of DNA was incubated in incubation buffer (0.75% SDS, 5% Triton-X-100, 750 mM NaCl, 5 mM EDTA, 2.5 mM EGTA, 100 mM Hepes, pH 7.4) with either 1 µg rabbit α-PfHP1 (for P. falciparum), 1 µg rabbit α-PvHP1 (for P. vivax and P. knowlesi) or 1 µg rabbit α-PbHP1 (for P. berghei, P. chaubaudi and P. yoelii) as well as 10 µl protA and 10 µl protG Dynabeads suspension (Life Technologies, #10008D and #10009D). For each sample four ChIP reactions were prepared and incubated overnight at 4°C while rotating. Beads were washed twice with wash buffer 1 (0.1% SDS, 0.1% DOC, 1% Triton-X100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes, pH 7.4), once with wash buffer 2 (0.1% SDS, 0.1% DOC, 1% Triton-X100, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes, pH 7.4), once with wash buffer 3 (250 mM LiCl, 0.5% DOC, 0.5% NP-40, 1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes, pH 7.4) and twice with wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, 20 mM Hepes, pH 7.4). Each wash was performed for 5 min at 4°C while rotating. Subsequently, immunoprecipitated chromatin was eluted in elution buffer (1% SDS, 0.1M NaHCO3) at room temperature. The eluted chromatin samples and the corresponding input samples (sonicated input chromatin containing 500 ng DNA) were de-crosslinked in 1% SDS/0.1 M NaHCO3/1 M NaCl at 45°C overnight while shaking. For each parasite strain or species the separate ChIP samples were combined and the DNA was purified using QIAquick MinElute PCR columns (Qiagen).
For each sequencing library 2-10 ng of ChIP or input DNA were end-repaired, extended with 3′ A-overhangs and ligated to barcoded NextFlex adapters (Bio Scientific, #514122) as described previously (Hoeijmakers et al., 2011). Libraries were amplified (98°C for 2 min; four cycles 98°C for 20 sec, 62°C for 3 min; 62°C for 5 min) using KAPA HiFi HotStart ready mix (KAPA Biosystems, KM2602) and NextFlex primer mix (Bio Scientific, #514122) as described (Kensche et al., 2016). 225-325 bp fragments (including the 125 bp NextFlex adapter) were size-selected using a 2% E-Gel Size Select agarose gel (Invitrogen, #G6610-02) and amplified by PCR for eight or ten cycles under the same condition as described above. Library purification and removal of adapter dimers was performed with Agencourt AMPure XP beads in a 1:1 library:beads ratio (Beckman Coulter, #A63880).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

P. vivax sample collection and chromatin preparation Eight clinical P. vivax isolates were collected from malaria patients receiving treatment at clinics run by the Shoklo Malaria Research Unit on the North Western border of Thailand. Five milliliters of whole blood were collected in lithium heparin collection tubes by venepuncture. After leukocyte depletion using non-woven fabric filters (Antoshin Pte Ltd) these samples were cryopreserved in glycerolyte 57 solution (Baxter Pte Ltd) and stored in liquid nitrogen (Borlon et al., 2012). Frozen samples were thawed and matured ex vivo for 40 hours as described (Borlon et al., 2012; Russell et al., 2011). The resulting P. vivax schizonts cultures were crosslinked at 37˚C for 10 min in presence of 1% formaldehyde (Sigma F8775) and subsequently the reactions were quenched by 0.125 M glycine. The crosslinked RBCs were centrifuged at 200 g for 5 min, supernatants were removed and the RBC pellets snap-frozen in liquid nitrogen. The eight samples were thawed and pooled and nuclei isolated by releasing parasites from infected RBCs using 0.05% saponin followed by lysis in CLB. Nuclei were washed and snap-frozen in CLB supplemented with 50% glycerol. Preparation of sheared chromatin was performed as described above for P. falciparum.
P.vivax-HP1_over_P.vivax-Input.BedGraph.gz

Data processing

RTA v2 software for base calling and bcl2fastq v2.16.0.10 conversion software for fastq conversion
Data were mapped with BWA samse (Version: 0.7.12-r1039) against the respective reference genome (P. berghei ANKA, P. chabaudi chabaudi, P. yoelii yoelii YM, P. falciparum 3D7 and P. knowlesi H from PlasmoDB version 26 and P. vivax P01 from PlasmoDB version 29. Pf2004 samples were additionally mapped against a draft Pf2004 genome assembly kindly provided by Thomas Otto (Welcome Trust Sanger Institute)
Sam files were converted to bam files and mapped reads were filtered to mapping quality ≥15 (SAM tools v1.2). Only uniquely mapped reads were used for further analysis.
BedGraph files were generated using bedtools genomecov with the option -bga and normalized using -scale and the scaling factor ‘1000000/amount of unique reads’.
Begraph log2 ratio were genereated using bedtools unionbedg
Genome_build: see second processing step
Supplementary_files_format_and_content: BedGraph log2 ratio files show the coverage as log2 ratio of ChIP over Input calculated using normalized BedGraph files

Submission date

Aug 15, 2017

Last update date

May 15, 2019

Contact name

Sabine Fraschka

Organization name

Radboud University Nijmegen

Department

Molecular Biology

Lab

Richárd Bártfai