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| Status |
Public on Jun 06, 2023 |
| Title |
ChIP-seq of Plasmodium berghei hepatic merozoites: H3K4me3 |
| Sample type |
SRA |
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| Source name |
ANKA
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| Organism |
Plasmodium berghei |
| Characteristics |
Stage: Hepatic merozoite
chip antibody: H3K4me3
strain: ANKA
isolation: HepG2 liver cells were cultured according to standard practices in complete DMEM medium (Dulbecco’s Modified Eagle Medium supplemented with 10% (v/v) fetal bovine serum, 1X glutaMAX, 100 U/ml penicillin, and 100 µg/ml streptomycin) at 37°C and 5% CO2. The day prior to infection, HepG2 cells were seeded at 75,000 cells per well in a volume of 500 µl of complete DMEM in a 24-well plate. The day of infection, salivary glands containing P. berghei sporozoites were dissected from live mosquitos (obtained from the University of Georgia insectary, GA, USA) and ground with a small pellet pestle to release sporozoites. Released sporozoites were filtered through a 40 µm filter and counted using a hemocytometer. Sporozoites were diluted to 40,000 sporozoites per 300 µl in infection medium (complete DMEM supplemented with 100 U/ml penicillin, 50 µg/ml streptomycin, and 150 µlg/ml neomycin and 50 µg/ml kanamycin, and 7.5 µg/ml of the antifungal amphotericin B. Culture medium was removed from the wells containing the adherent HepG2 cells and 300 µl of the sporozoite dilution was added to each well. The plate was centrifuged at 2,000 × g for 5 min. at RT. Two hours post infection, all medium was removed and replaced with 1 ml infection medium. At 48 hpi the medium replaced once more with 1 ml fresh infection medium. Infected cells were cultured under normal conditions until merosomes were harvested 68 – 75.5 hpi. To harvest merosomes, the culture medium was collected in a 15-ml tube. Next, the empty wells were rinsed with 500 µl of infection medium and this medium was added to the original collection. Merosomes were pelleted by centrifugation at 4,000 × g for 5 min. at RT. Supernatant was removed until roughly 100 µl remained. The merosome pellet was resuspended in the remaining supernatant and transferred to a 1.5-ml tube. PBS was added to the merosomes to a final volume of 1 ml. Merozoites were released from merosomes by incubation with 1% saponin in milliQ water for 2 min. at RT. Merozoites were pelleted by centrifugation at 4,000 × g for 4 min. at RT. Supernatant was removed until roughly 100 µl remained, and merozoites were resuspended in the remaining medium. PBS was added to the resuspended merozoites to a final volume of 1 ml.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Parasites were crosslinked with 1% paraformaldehyde. Crosslinked parasites were washed with 1 ml cold PBS containing protease inhibitors (cOmplete protease inhibitor cocktail) then incubated on ice with 1 ml nuclear extraction buffer [f.c.: 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 200 mM AEBSF, cOmplete protease inhibitor cocktail, Phos Stop phosphatase inhibitor in nuclease-free water]. After 35 min., NP-40/IGEPAL was added to a final concentration of 0.25% and the solution was homogenized by passing the mixture seven times through a 26 G needle. Extracted nuclei were pelleted by centrifugation at 2,100 × g for 20 min. at 4°C. Supernatant was removed and the pelleted nuclei were resuspended in 130 µl of cold shearing buffer [f.c.: 0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 7.5, cOmplete protease inhibitor cocktail, Phos Stop phosphatase inhibitor in nuclease-free water] and transferred to a microTUBE. Chromatin was fragmented with an M220 sonicator (Covaris) with the following settings: 5% duty cycle, 75 W peak incident power, 200 cycles per burst at 6°C for 300 sec. Fragmented chromatin was transferred to a pre-lubricated, low binding 1.5 ml tube. The microTUBE was washed with 70 µl of shearing buffer after which the solution was combined with the fragmented chromatin for a total volume of 200 µl. For each developmental stage (schizont, merozoite, ring, and hepatic merozoite) 30 µl of sheared chromatin was collected at this point for use as an input sample and stored at -70°C until decrosslinking. Chromatin was diluted 1:1 in ChIP dilution buffer [f.c.: 30 mM Tris-HCl pH 8.0, 3 mM EDTA, 0.1% SDS, 300 mM NaCl, Triton X-100, supplemented with cOmplete protease inhibitor cocktail and Phos Stop phosphatase inhibitor in nuclease-free water] then precleared by incubation with protein A agarose/salmon sperm DNA beads (50 µl beads per 1 ml sheared chromatin) for 1 hour at 4°C with agitation to reduce non-specific background. Following the incubation, the beads were pelleted by centrifugation at 100 × g for 1 min. at RT. The supernatant containing the chromatin was moved to a new low-binding tube and incubated overnight at 4°C with agitation with 2 µg of antibody against the histone PTM of interest. Protein A agarose/salmon sperm DNA beads (25 µl beads per 500 µl sonicated chromatin) were washed with 500 µl ChIP dilution buffer and blocked with 1 mg/ml BSA for 1 hour at 4°C with agitation to reduce non-specific binding. Agarose beads were washed 3 times with 500 µl ChIP dilution buffer, then added to the chromatin suspension and incubated for 1 hour at 4°C with agitation. Agarose beads were pelleted by centrifugation at 100 × g for 1 min. at RT, and the supernatant was discarded. Beads were washed twice at 4°C for 15 min. with agitation 1 ml high salt wash buffer [f.c.: 1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 nM NaCl in nuclease-free water], 1 ml low salt wash buffer [f.c.: 1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, in nuclease-free water], and 1 ml lithium chloride wash buffer [f.c.: 0.25 M LiCl, 1% NP-40/IGEPAL, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0 in nuclease-free water]. Beads were then washed twice for 15 min. with TE buffer [f.c.: 10 mM Tris-HCl pH 8.0, 1 mM EDTA in nuclease-free water] at RT with agitation. Chromatin bound to agarose beads was eluted by incubating with 125 µl elution buffer [f.c.: 1% SDS, 0.1 M NaHCO3 in nuclease-free water] for 15 min. at RT with agitation. The elution was repeated, and fractions combined for a total of 250 µl. Samples were reverse crosslinked by adding NaCl to a final concentration of 0.5 M and incubating overnight at 45°C. RNase A was then added to a final concentration of 0.6 μg/ml and incubated at 37°C for 30 min. Samples were then incubated with EDTA (f.c.: 1 mM), Tris-HCl pH 7.5 (f.c.:4 mM), and proteinase K (0.8 U) for 2 hours at 45°C. DNA was isolated using 2 volumes sparQ beads according to manufacturer’s instructions. ChIP DNA was stored at -20°C until library preparation. Input samples were incubated with 0.6 μg RNase A per µl for 30 min. at 37°C, then incubated with 0.8 U proteinase K overnight at 45°C. DNA was isolated using 2 volumes sparQ beads according to manufacturer’s instructions and stored at -20°C until library preparation.
Libraries from ChIP samples were prepared using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB #E6040) or the NEBNext Ultra II DNA library prep kit for Illumina (NEB #E7645) according to manufacturer’s instructions with the following modification. Libraries were amplified using i7 index primers and i5 universal primer (NEBNext multiplex oligos for Illumina index primers set 1 and 2, NEB #E7335 and #E7500) with KAPA HiFi hot start ready mix with the following program: initial denaturation at 98°C for 45 sec.; 15 cycles of (98°C for 15 sec., 55°C for 30 sec., 62°C for 30 sec.); and final extension at 62°C for 5 min. Libraries were purified using 0.9 volumes sparQ beads according to manufacturer’s instructions. Libraires were sequenced with the Illumina HiSeq 2500 or Illumina NextSeq500 to generate 50 bp single-end reads or 75 bp paired-end reads, respectively.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Pb H3K4me3
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| Data processing |
Paired-end data generated for histone PTM ChIP-seq was treated as single-end data so that all ChIP-seq data could be processed in the same way, and only read one was processed for analysis. Following quality assessment of the reads using FastQC (v0.11.8) (reads with a quality score ³ 30 were retained), adaptor sequences were trimmed using Trim-galore (v0.5.0). ChIP-seq data were mapped to the P. falciparum 3D7 genome v3.0, release 44 or the P. berghei ANKA genome v3.0, release 49 with Bowtie2 (v2.3.5). Reads were filtered with SAMtools (v1.9) to remove sequences that aligned to the genome more than once (option -q 10) and duplicates were removed using markduplicates from Picard (v2.18.29-SNAPSHOT). Using bamCompare from the deeptools suite (v3.2.0), log2(ChIP/input) bigwig files with reads extended to 150 bp were generated from bam files. Input and no antibody bigwig files were generated with bamCoverage from deeptools (v3.2.0).
Assembly: P. falciparum 3D7 genome v3.0, release 44 or P. berghei ANKA genome v3.0, release 49
Supplementary files format and content: bigwig files showing log2(ChIP/input), except for input and no antibody samples which not normalized
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| Submission date |
Oct 13, 2022 |
| Last update date |
Jun 06, 2023 |
| Contact name |
Evelien M Bunnik |
| Organization name |
University of Texas Health Science Center at San Antonio
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| Department |
Microbiology, Immunology, and Molecular Genetics
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| Street address |
7703 Floyd Curl Dr.
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| City |
San Antonio |
| State/province |
TX |
| ZIP/Postal code |
78229 |
| Country |
USA |
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| Platform ID |
GPL23133 |
| Series (2) |
| GSE215426 |
Histone post translation modification differences during the Plasmodium schizont-to-ring transition [ChIP-seq] |
| GSE215429 |
Histone post translation modification and Transcriptional differences during the Plasmodium schizont-to-ring transition |
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| Relations |
| BioSample |
SAMN31271695 |
| SRA |
SRX17878916 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6636926_log2ratio_merosomes_H3K4me3_to_input_R1_072022_ext.bw |
13.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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