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Sample GSM2743113

Query DataSets for GSM2743113

Status

Public on Jan 16, 2018

Title

Pf-3D7-Trophozoite-HP1

Sample type

SRA

Source name

intraerythrocytic stage, trophozoite stage

Organism

Plasmodium falciparum

Characteristics

strain: 3D7
antibody: alpha-PfHP1

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. falciparum sample collection and chromatin preparation P. falciparum parasites were cultured at 5% haematocrit based on the original protocol published by Trager and Jensen (Trager and Jensen, 1978). Growth synchronization was achieved by repeated sorbitol treatments (Lambros and Vanderberg, 1979). 3D7 parasites were cultivated with AB+ human RBCs in RPMI 1640/25 mM Hepes standard culture medium supplemented with 0.5% Albumax II. NF54 and NF135 parasites were cultivated with O+ human RBCs in RPMI 1640/25 mM Hepes standard culture medium supplemented with 10 % human serum. Pf2004/164tdTom parasites (Brancucci et al., 2015) were cultivated with AB+ human RBCs in RPMI 1640/25 mM Hepes standard culture medium supplemented with 10 % human serum. Pf2004/164tdTom gametocytes were generated by inducing sexual commitment as described (Brancucci et al., 2015). After re-invasion cultures were treated with 50mM N-acetylglucosamine (Fivelman et al., 2007) for three consecutive days to eliminate asexual parasites. Chromatin from 3D7 ring stages, trophozoites or schizonts (approximately 0.75-1.5x109 parasites each) and from Pf2004/T164dTom schizonts, stage II/III gametocytes (day four after re-invasion) or stage IV/V gametocytes (day nine after re-invasion) (approximately 2x108 parasites each) was prepared by crosslinking cultures with 1% formaldehyde (Sigma F8775) for 15 min at 37ºC. Crosslinking reactions were quenched by 0.125 M glycine. Nuclei were isolated by releasing parasites from infected RBCs using 0.05% saponin followed by lysis in CLB (20 mM Hepes, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 0.65% NP-40, 1mM DTT, 1x protease inhibitor (Roche Diagnostics), pH 7.9). Nuclei were washed and snap-frozen in CLB supplemented with 50% glycerol. Chromatin from NF54 and NF135 parasites (approximately 2-4x109 parasites each) was prepared by passing the cultures through Plasmodipure filters (EuroProxima) to remove white blood cells prior to formaldehyde crosslinking for 10 min at 37ºC and quenching in 0.125 M glycine. Nuclei were isolated by releasing parasites from infected RBCs using 0.05% saponin followed by gentle homogenisation (pestle B, 15 strokes) in CLB2 (10 mM Tris-HCl, 3 mM MgCl2, 0.2% NP40, 1x protease inhibitor (Roche Diagnostics), pH 8.0) and centrifugation through a 0.25 M sucrose cushion (in CLB2) at 2000 rpm for 10 min at 4°C. Nuclei were snap-frozen in CLB2 supplemented with 20% glycerol. Frozen nuclei were thawed and resuspended in sonication buffer (50mM Tris-HCl, 1% SDS, 10mM EDTA, 1x protease inhibitor (Roche Diagnostics), pH 8.0) and sonicated for 20-24 cycles of 30 sec ON/30 sec OFF (setting high, BioruptorTM Next Gen, Diagenode). Chromatin fragment sizes ranged from 100-600 bp as determined by de-crosslinking a 50 l aliquot and running the purified DNA on a 1% agarose gel
Pf-3D7-Trophozoite-HP1_over_Pf-3D7-Schizont-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