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Sample GSM2491675

Query DataSets for GSM2491675

Status

Public on May 04, 2018

Title

GDVDD_2h_OFF_GFP

Sample type

SRA

Source name

red blood cell stage, schizont

Organism

Plasmodium falciparum

Characteristics

developmental stage: red blood cell stage, schizont
strain: 3D7
genomic modification: episomal GDV1-GFP-DD construct, driven by calmodulin promoter

Treatment protocol

Parasite cultures were synchronized by repeated sorbitol treatments (Lambros and Vanderberg, 1979).

Growth protocol

Parasite-infected red blood cells were cultured in petri dishes at 5% hematocrit in the presence of 3% O2 and 4% CO2 (rest N2) in RPMI 1640 supplemented with 25mM Hepes, 50mg/l Hypoxanthine, 0.21% NaHCO3, 0.5% Albumax and 50mg/l Neomycin sulphate. The 3D7/GDV1-GFP-DD parasite line was cultured in presence of 4nM WR99210 (WR).

Extracted molecule

genomic DNA

Extraction protocol

Synchronized 3D7/GDV1-GFP-DD schizonts on and off Shield1 at 5% parasitemia were cross-linked by incubation with a final concentration of 1% formaldehyde for 15 min at 37ºC. The cross-linking reaction was quenched by addition of 0.125M glycine (final concentration). Nuclei were isolated by releasing parasites from iRBCs using 0.05% saponin followed by lysis in CLB (20 mM Hepes, 10mM KCl, 1mM EDTA, 1mM EGTA, 0.65% NP-40, 1mM DTT supplemented with protease inhibitor). Nuclei were washed and snap-frozen in CLB supplemented with 50% glycerol. To obtain fragmented chromatin for ChIP, nuclei were resuspended in sonication buffer (50 mM Tris pH:8.0, 1% SDS, 10mM EDTA, supplemented with protease inhibitor) and sonicated for 20 cycles of 30 sec ON/30 sec OFF (setting high, BioruptorTM, Diagenode). Fragment sizes ranged from 200-600 bp as determined by decrosslinking a 50μl aliquot and running the purified DNA on a 1.5% agarose gel. ChIPs were performed by incubating ~500 ng (DNA-content) sonicated chromatin from 3D7/GDV1-GFP-DD on and off Shield1 overnight while rotating at 4°C with 1 μg mouse anti-GFP (Roche Diagnostics, #11814460001) or 1 μg rabbit anti-PfHP1 (Brancucci et al. 2014), respectively, in incubation buffer (5% Triton-X-100, 750mM NaCl, 5mM EDTA, 2.5mM EGTA, 100mM Hepes pH 7.6) with 10 μl protA and 10 μl protG Dynabeads (Life Technologies, 10008D and 10009D). Beads were washed for 5 min at 4°C while rotating with 400 μl of the following wash buffers: 2x wash buffer 1 (0.1% SDS, 0.1% DOC, 1.0% Triton-X100, 1mM EDTA, 0.5mM EGTA, 20mM Hepes pH 7.6), 1x wash buffer 2 (0.1% SDS, 0.1% DOC, 1.0% Triton-X100, 500mM NaCl, 1mM EDTA, 0.5mM EGTA, 20 mM Hepes pH 7.6), 1x wash buffer 3 (250mM LiCl, 0.5% DOC, 0.5% NP-40, 1mM EDTA, 0.5mM EGTA, 20mM Hepes pH7.6), 2x wash buffer 4 (1mM EDTA, 0.5mM EGTA, 20 mM Hepes pH7.6). The immunoprecipitated chromatin was eluted in 200 μl elution buffer (1% SDS, 0.1M NaHCO3) while rotating for 20 min at RT and decrosslinked in decrosslinking buffer (1%SDS, 0.1M NaHCO3, 1M NaCl) in a 45°C shaking heat-block overnight. In parallel, 30 μl of sonicated input chromatin was decrosslinked under the same conditions. The DNA was purified over QIAquick MinElute PCR columns (Qiagen) and eight separate anti-GFP or two anti PfHP1ChIPs were pooled over one column.
For each sequencing library generated from 3D7/GDV1-GFP-DD parasites 1ng (anti-mouse GFP antibody) , 5ng (anti-HP1 antibody) of ChIP DNA or 1 ng of the input DNA were end repaired, extended with 3′ A-overhangs, and ligated to barcoded NextFlex adapters (Bio Scientific, #514122). Libraries were amplified using the KAPA HiFi HotStart ready mix (KAPA Biosystems) and NextFlex primer mix (Bio Scientific, #514122) as follows: 98 °C for 2 min; 4 cycles of 98 °C for 20 sec, 62 °C for 3 min; 62 °C for 5 min. Amplified libraries were gel size-selected for 225-325 bp (mono-nucleosomes + ~125 bp NextFlex adapter) using 2% E-Gel Size Select agarose gels (Invitrogen, #G6610-02) and again amplified as described above for 10 cycles. Libraries were purified and adapter dimers removed by Agencourt AMPure XP beads purification using a1:1 library beads ratio (Beckman Coulter, #A63880).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

chip antibody: anti-mouse GFP antibody (Roche Diagnostics, #11814460001)
anti-GFP ChIP

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) to the P. falciparum 3D7 reference genome from PlasmoDB version 26 (http://www.plasmodb.org).
Sam files were converted to bam files, q15 filtered and filtered for unique reads
Bedgraph files were generated using bedtools genomecov with the option –bga (regions with zero coverage are also reported) and normalized using the option -scale and the scaling factor ‘1000000/amount of unique reads’.
Begraph log2 ratio or substraction files were genereated using bedtools unionbedg
Genome_build: PlasmoDB Plasmodium falciparum 3D7 release 26
Supplementary_files_format_and_content: Bedgraph log2 ratio files show the coverage as log2 ratio of ChIP over Input calculated using normalized Bedgraph files
Supplementary_files_format_and_content: Bedgraph substraction files show the coverage as substraction of GDVDD ON ChIP minus GDVDD OFF ChIP calculated using the normalized Begraph files

Submission date

Feb 14, 2017

Last update date

May 15, 2019

Contact name

Sabine Fraschka

Organization name

Radboud University Nijmegen

Department

Molecular Biology

Lab

Richárd Bártfai