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| Status |
Public on Jul 01, 2023 |
| Title |
H3K4me3, +MC+3DZA, repA (Sample 18) |
| Sample type |
SRA |
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| Source name |
asexual blood stages
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| Organism |
Plasmodium falciparum |
| Characteristics |
strain: Pf-peg4-tdTomato
developmental stage: asexual blood stages
chip antibody: H3K4me3 antibody ChIP Grade (polyclonal) Rabbit IgG, Diagenode Catalog #C15410003-50
treatment: +Met+Cho+3DZA
cell type: schizonts
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| Treatment protocol |
Synchronous P. falciparum parasites were grown from 8hpi to 34hpi ± 6 hpi (100mL, 3% hematocrit, 5-6% parasitemia) under the indicated growth conditions. Infected red blood cells (iRBC) were then purified to > 90% purity using a 70/40% percoll/sorbitol density gradient and centrifugation (4700 G for 15 minutes at room temperature), then washed three times with minimal RPMI. Following isolation, equal numbers of isolated iRBC, as determined by the Beckman Coulter Z1 Coulter Particle Counter, were then re-incubated in their respective treatment conditions for four more hours to allow parasites to recover from percoll/sorbitol isolation. Following recovery, the purity of iRBCs was assessed using flow cytometry,
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| Growth protocol |
The parasite strains used for this study were NF54 obtained from BEI Resources and the Pf-peg4-tdTomato 43. Cultures were maintained using established culturing techniques. Standard complete media was RPMI-1640 supplemented with 25 mM HEPES, 368 μM hypoxanthine, 1 mM sodium hydroxide, 24 mM sodium bicarbonate, 21 μM gentamycin (Millipore Sigma), with Albumax II Lipid-Rich BSA (Gibco), unless otherwise stated. Concentrations for choline and lysoPC supplementation were used according to 14. See Supplementary Table 1 for changes to media compositions used throughout this manuscript. RBCs or cultures were washed three times with respective experimental media conditions at the start of each change in media condition
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
H3K9me3 and H3K4me3 abundances were measured by Cleavage Under Targets & Release Using Nuclease (CUT & RUN) 46 adapted for P. falciparum. Approximately 1 x 10^7 percoll/sorbitol isolated schizonts (34-38 hpi) per sample were washed three times with 1 ml wash buffer (20 mM HEPES at pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and 1 × Roche complete protease inhibitor) at room temperature and resuspended in 225ul of wash buffer. 25ul of Concanavalin A-coated beads were washed and resuspended in binding buffer (20 mM HEPES–KOH at pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) before being added to each sample. Samples were then rotated for 10 minutes at room temperature. Then, samples were placed on a magnetic stand to clear (30s to 2 minutes) and remove all the liquid. Samples were washed 3x’s with DIG wash buffer (wash buffer with 0.025% digitonin). For each sample, 150μl of antibody wash buffer was added (wash buffer, 0.025% digitonin, and 2 mM EDTA at pH 8.0) with H3K4me3 (0.005μg/μl, C15410003-50, Diagenode), H3K9me3 (0.005μg/μl, ab8898, abcam), or IgG isotype control (0.005μg/μl, 02-6102, ThermoFisher) was added to the sample tube and incubated at 4 °C, rotating, overnight. Following antibody incubation, samples were placed on a magnetic stand to clear and pull off the liquid, then washed 3 x’s with DIG wash buffer, 150 μl of ProteinA/G-MNASE fusion protein (dilution 1:60, 15-1016, EpiCypher) in DIG wash buffer and rotated at 4 °C for 1 hour. After washing samples 3x’s with DIG wash buffer, samples were then washed 3x’s with Low Salt Rinse Buffer (20 mM HEPES–NaOH at pH 7.5, 0.025% digitonin, 0.5 mM spermidine). 200 μl of ice-cold incubation buffer (3.5 mM HEPES–NaOH at pH 7.5, 100mM CaCl2, 0.025% DIG) was added and samples were equilibrated at 0 °C for 30 minutes to achieve targeted digestion. Digestion was then stopped by adding 200 μl of stop buffer (170 mM NaCl, 20 mM EGTA at pH 8.0, 20 mM EGTA, 0.0.25% digitonin, 25 μg/mL glycogen, 50 μg/mL RNase A). After incubation at 37 °C for 30 minutes to release soluble fragments, samples were digested by adding 2.5 μL proteinase K (20 mg/ml) and 2 μL 10% SDS, and then incubated at 50 °C for 1 hour. DNA fragments were purified with phenol–chloroform–isoamyl alcohol and washed by ethanol precipitation, and finally dissolved in 30 μL TE buffer (1mM Tris-HCl pH 8, 0.1 mM EDTA).
Library construction was carried out using NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S, NEB). 4ng of fragmented DNA was used for end-repair/A-tailing, ligation, and post ligation cleanup with 1.7x volumes of AMPure XP beads (Catalog number, company). Following cleanup, PtdChoR amplification was performed using 2x KAPA HotStart ready mix (Catalog number, company) and NETFLEX primer mix (Catalog number, Bio Scientific) with PtdChoR program: 1 min @ 98o C/15 cycles: 10 sec @ 98o C/1 min @ 65o C// 5min @ 65o C// hold 4o C. PtdChoR products were size selected with 0.8x volumes, then 1.2x volumes of AMPure XP beads. Beads were then washed twice with 80% ethanol and DNA eluted with 0.1x TE and used for sequencing in a Nextseq2000 system as paired-end reads, following quality control.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Library strategy: CUT&RUN
After sequencing, raw reads were trimmed using Trimmomatic v0.38 47 to remove residual adapter sequences and low quality leading and trailing bases. Both paired and unpaired reads were retained for read lengths that were at least 30 bases after trimming. Trimmed paired reads were aligned to the PlasmoDB version 46 P. falciparum 3D7 reference genome 26 using BWA v.0.7.1 48. SAMtools v.1.10 49 was used to remove low quality alignments as well as sort and index sample files. Normalized fold enrichment tracks were generated by using the MACS2 v.2.2.7.1 50 callpeak function with settings: -f BAMPE -B -g 2.3e7 -q 0.05 –nomodel –broad –keep-dup auto –max gap 500. Bedgraph outputs were then passed into the bdgcmp function with the setting -m FE (fold enrichment) to generate signal tracks to profile histone modification enrichment levels compared to whole genome. Peak sets from replicates were compared with Bedtools intersect v2.26.0 51, and peaks that overlapped by at least 1 bp were considered shared. Fold enrichment bedgraphs and peak sets were then output to RStudio Server (v1.4.1717) for further analysis using the GenomicRanges Package v.1.44.0 52 and visualized with the GViz v1.38.1 package 53 within the Bioconductor project (release 3.13) 54
Genome_build: Plasmodium falciparum 3D7 v51 from PlasmoDB
Supplementary_files_format_and_content: bedgraph, bed
X*.bedgraph files (bedgraph of enrichment foldchange versus matched IgG control)
delta*.bedgraph files (bedgraph of difference in K9 enrichment)
bed file of called peaks
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| Submission date |
Mar 04, 2022 |
| Last update date |
Jul 01, 2023 |
| Contact name |
Bjorn FC Kafsack |
| Organization name |
Weill Cornell Medicine
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| Department |
Microbiology & Immunology
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| Street address |
1300 York Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL32020 |
| Series (1) |
| GSE197916 |
Metabolic competition between lipid metabolism and histone methylation regulates sexual differentiation in human malaria parasites |
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| Relations |
| BioSample |
SAMN26434106 |
| SRA |
SRX14366838 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5932341_X3.S7.X3.NF54.MCDZA.K4.A_SPMR_FE_comp.bedgraph.gz |
101.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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