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| Status |
Public on Aug 07, 2023 |
| Title |
Input_20R_rep2 |
| Sample type |
SRA |
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| Source name |
P. falciparum
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| Organism |
Plasmodium falciparum |
| Characteristics |
chip antibody: input
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| Growth protocol |
Parasites were maintained in human A+ erythrocytes at 37 °C in RPMI 1640 medium containing AlbumaxII (Invitrogen) supplemented with 2 mM L-glutamine. Parasites were either synchronized by sorbitol treatment or by purifying mature schizont stages using 70% (v/v) isotonic Percoll (GE Healthcare Life Sciences) before allowing reinvasion to occur, followed by sorbitol treatment.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The ChIP assay was performed as described (Pandey et al., 2020) with a few modifications. Parasite culture (50 ml) containing synchronized late-stage schizonts (~ 40 h.p.i.) at ~5% parasitemia was centrifuged at 900 g for 4 min and the cells washed once with PBS. To the cell pellet, 25 ml of 0.15% saponin in PBS was added and incubated on ice for 10 min followed by washing twice with cold PBS. Parasites were crosslinked for 10 min by adding methanol-free formaldehyde at 1% final concentration and incubated for 10 min at 37 °C with occasional shaking. The crosslinking reaction was quenched by adding 1.25 M glycine to achieve a final concentration of 0.125 M and incubated at 37 °C for another 5 min. Parasites were centrifuged for 10 min at 3250 g at 4 °C, washed three times with DPBS, snap frozen in liquid nitrogen and stored at -80 °C until further use. Frozen formaldehyde fixed parasites were thawed on ice for chromatin immunoprecipitation. One ml of nuclear extraction buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1mM DTT, 1x EDTA-free protease inhibitor cocktail [Roche]) was added to the tubes containing thawed parasites and incubated on ice for 30 min. After the incubation, 10% NP-40 was added to reach a final concentration of 0.25% and the parasites were lysed by passing through a 261/2 G needle seven times. Parasite nuclei were collected by centrifuging at 2500g for 10 min at 4 °C. Shearing of chromatin was carried out using the Covaris Ultra Sonicator (E220) for 14 min with the following settings; 5% duty cycle, 140 intensity peak incident power, 200 cycle per burst to obtain fragment size of 200 to 600 bp. Insoluble materials were removed by centrifuging the sheared chromatin for 10 min at 13500 g at 4 °C. 30 µl of fragmented chromatin were stored as input at -80°C. Fragmented chromatin was diluted 1:1 in ChIP dilution buffer (30 mM Tris-HCl pH 8.0, 0.1% SDS, 3 mM EDTA, 300 mM NaCl, 1% Triton X-100, EDTA-free protease inhibitor cocktail). Chromatin was incubated overnight with 6 µg of mentioned antibodies. The antibody-protein complex was recovered with protein A coupled to magnetic beads (Dynabeads, Invitrogen, Cat No. 10002D), followed by extensive washes with low salt immune complex wash buffer, high salt immune complex wash buffer (washes done at 4 °C) and TE buffer (washes done at RT). Chromatin was eluted with elution buffer (1% SDS, 0.1 M NaHCO3) at 45 °C for 30 min with shaking. Immunoprecipitated chromatin and input were reverse crosslinked overnight at 45 °C by adding 5 M NaCl to a final concentration of 0.5 M. Samples were treated with RNaseA for 30 min at 37 °C followed by a 2 h incubation at 45 °C with proteinase K (final concentration 66 µg/ml). DNA was purified using ChIP DNA clean & concentrator (Zymo Research, Cat. No. D5205).
Libraries were prepared using NEBNext Ultra II DNA library kit following the manufacturer’s instructions until the step of adapter ligation (Adapters were diluted at 1: 20 ratio). Adapter ligated libraries were purified using AmpureXP beads. The libraries were amplified for a total of 6 PCR cycles (2 min at 98 °C initial denaturation; 6 cycles of 30 s at 98 °C, 50 s at 62 °C, final extension 5 min at 62 °C) using the KAPA HiFi HotStart Ready Mix (Kapa Biosystems). Amplified libraries were purified and size selected for 350 bp inserts using AmpureXP beads and sequenced on the Illumina HiSeqX platform with 150 bp paired-end read layouts.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
bcl2fastq2 Conversion Software
Low-quality reads and Illumina adaptor sequences from the read ends were removed using Trimmomatic (Bolger et al., 2014). Quality trimmed reads were aligned to the P. falciparum genome (plasmodb.org, v3, release v32) using HiSat2. Duplicate reads were removed using samtools (markdup) (Li et al., 2009). GC bias was corrected using deeptool’s correctGCBias tool (Ramirez et al., 2014). For coverage plots of Api2EI 40 h.p.i. and 20 h.p.i. ChIP-seq experiments, deeptool’s bamCompare tool was used to normalize the read coverage per base of the genome position ( option ‘-bs 1’) in the respective input and ChIP samples to the total number of reads in each library (-- nomarmalizeUsing RPKM). Normalized input coverage per bin was subtracted from the ChIP values (option – operation subtract). Coverage plots were visualized using IGV genome browser (Thorvaldsdottir et al., 2013).
ChIP-Peaks (q-value cutoff < 0.05) were identified using macs2 (Zhang et al., 2008) by comparing the input with ChIP with default settings but without prior peak modelling (option ‘-nomodel’), the fragment size set to 200 bp (option ‘-extsize 200’) and the genome size (option ‘-g’) set to 233332839. Robust common peaks between replicates were identified using bedtools ‘intersect’ (option –f 0.30 –r) (Quinlan and Hall, 2010). Peak annotation was carried out using Homer’s annotatePeaks.pl that assigned each peak with the nearest downstream gene. Enrichment heatmaps and profile plots were generated using the deepTools computeMatrix and plotHeatmap tools.
Assembly: Plasmodium falciparum build version 3
Supplementary files format and content: Bam files were converted to bigwig files using bamCoverage from the deeptools suite
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| Submission date |
Apr 20, 2023 |
| Last update date |
Aug 07, 2023 |
| Contact name |
AMIT KUMAR SUBUDHI |
| E-mail(s) |
amit.subudhi@kaust.edu.sa
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| Phone |
+96540375986
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| Organization name |
King Abdullah University of Science and Technology
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| Department |
BESE
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| Lab |
Pathogen Genomics
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| Street address |
Level4, Builiding, 2, KAUST
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| City |
Thuwal |
| State/province |
Thuwal |
| ZIP/Postal code |
239556900 |
| Country |
Saudi Arabia |
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| Platform ID |
GPL26836 |
| Series (2) |
| GSE190519 |
PfAP2-P DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages |
| GSE230206 |
PfAP2-P DNA-binding protein is a master regulator of parasite pathogenesis during malaria parasite blood stages (ChIP-Seq II) |
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| Relations |
| BioSample |
SAMN34271727 |
| SRA |
SRX20030231 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7192262_Input_20R_rep2.bw |
8.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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