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| Status |
Public on May 07, 2022 |
| Title |
D6F, Input, rep3 |
| Sample type |
SRA |
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| Source name |
cell culture
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| Organism |
Plasmodium falciparum |
| Characteristics |
parasite line: 3D7-ABCG2-GFP
sort strategy: GFP high, no mitotracker
Sex: female
chip antibody: none
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| Treatment protocol |
Gametocytogenesis was induced by stressing the parasites through lowering the hematocrit and cultivation in partially spent medium. Asexual parasites were selectively inhibited by addition of N-acetyl-D-glucosamine, from day 0 to day 6 of gametocytogenesis. Gametocytes were enriched magnetically on a MACS CS column on day 3 and FACS sorted using GFP only or GFP and Mitrotracker Deep Red (as defined below) directly before nuclei were harvested. Asexual parasites were not sorted.
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| Growth protocol |
3D7::ABCG2-GFP P. falciparum parasites were cultivated at 5 % haematocrit in complete culture medium (RPMI-1640 complete medium (Gibco, Thermo Fisher Scientific) complemented with 25 mM HEPES pH 7.3 and 20 µg/ml gentamycin (Thermo Fisher Scientific) and supplemented with 0.25 % AlbuMAX II Lipid-Rich BSA (Thermo Fisher Scientific) and 5 % human AB Rh+ serum purchased from the Bavarian Red Cross Service (BRK). The parasites were propagated in erythrocytes of blood group 0+ (purchased from BRK) in a gas environment of 1 % O2, 5 % CO2, and 94 % N2 at 37 °C. Developing gametocytes were magnetically purified using MACS columns and separated by sex using flow cytometry. Male gametocytes were GFP low and female gametocytes GFP high. Mitotracker was used to sort for viable cells.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Parasites were crosslinked at a final concentration of 1% PFA for 10 min at 37°C and subsequently quenched with 125 mM glycine for 5 min on ice. The cells were then spun down and lysed using 0.075% saponin in PBS. After lysis the cells were washed twice with PBS and then resuspended in cold lysisbuffer (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA pH 8.0, 0.1 mM EGTA pH 8.0, 1 mM DTT, 1x Protease Inhibitor (Roche); 1ml per 5*10^7 cells). The mix was then transferred to a tissue grinder and incubated on ice for 30 min. Then Igepal was added to a final concentration of 0.25% and cells were lysed with 100 strokes. The resulting free nuclei were again pelleted and lysed by SDS lysis buffer (1 % SDS, 10 mM EDTA, 50 mM Tris pH 8.1, 1x Protease Inhibitor). The DNA was then sheared by sonication (2 times 8 min with 30s ON 30s OFF cycles). The sheared material was spun and the supernatant was transferred and diluted 1:10 with ChIP dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 150 mM NaCl). The diluted chromatin was then precleared by BSA-blocked agarose G beads (GE Healthcare) for 1h. Subsequently 50µl material were set aside as input. For the IP 15µl agarose G beads were used per antibody per sample. The IP was incubated under agitation at 4°C over night and thereafter washed sequentially with different buffers: Low Salt Wash Buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 150 mM NaCl), then High Salt Wash Buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, 500 mM NaCl), then LiCl Wash Buffer (0.25 M LiCl, 1 % Igepal, 1 % Sodium-Desoxycholat, 1 mM EDTA, 10 mM Tris-HCl pH 8.1), then twice at RT with TE (10 mM Tris-HCl pH 8, 1 mM EDTA pH 8). The immunoprecipitated chromatin was eluted from the beads with 100 µl Elution Buffer (2 % SDS, 200 mM NaHCO3) for 15 min at RT, twice. The Input sample was also diluted to the same volume with Elution Buffer. 500 mM NaCl were added for decrosslinking (45 °C over night). Finally, the proteins were digested by addition of 2 µl Proteinase K (Thermo Fisher Scientific) for 1 h at 37 °C, and DNA was purified using the MinElute Kit (QIAGEN 28006).
Libraries were generated using the Accel-NGS (TM) 2S Plus DNA Library Kit for Illumina platforms (SWI Swift Bioscience) and Accel-NGS (TM) 2S Indexing Kit (SWI Swift Bioscience) according to the manufacturer's guidelines. AmpureXP beads (Beckman Coulter) were used for the purification steps and the amplification step was performed with 15 cycles using the KAPA Hifi PCR kit (KK2101; Roche) according to published procedures (PMID: 26067602).
directional paired-end ChIP-Seq
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Quality control of samples using FastQC version 0.11.8
Read trimming using TrimGalore 0.6.4, and if necessary Cutadapt 2.8 and again FastQC
Read alignment using Bowtie2 version 2.3.4.3 default settings
generation of bam files and merging of files by samtools 1.9
generation of bigwig files and correlation plots by Deeptools 3.1.2
visualisation by IGV 2.6.3 and pyGenometracks 3.6
peak calling using MACS2 2.1.2.1
further data analysis by bedtools 2.28.0, mixtools 1.2.0, ggplot2 3.3.2, scanpy 1.8.1
Assembly: PlasmoDB-28_Pfalciparum3D7_Genome.fasta / PlasmoDB-28_Pfalciparum3D7.gff (available on PlasmoDB)
Supplementary files format and content: bigwigfiles filtered by quality > 28, normalized using RPKM
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| Submission date |
May 04, 2022 |
| Last update date |
May 08, 2022 |
| Contact name |
Michaela Petter |
| E-mail(s) |
michaela.petter@uk-erlangen.de
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| Phone |
+4991318522177
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| Organization name |
Univeritätsklinikum Erlangen
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| Department |
Mirkobiologisches Institut
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| Street address |
Wassertrumstr. 3-5
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| City |
Erlangen |
| ZIP/Postal code |
91054 |
| Country |
Germany |
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| Platform ID |
GPL21078 |
| Series (1) |
| GSE202214 |
ChIP-Seq analysis of day 6 male and female gametocytes of the malaria parasite Plasmodium falciparum |
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| Relations |
| BioSample |
SAMN28090286 |
| SRA |
SRX15152534 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6106386_D6_1_CC65DANXX_GCCAAT_rpkm_ndq.bw |
2.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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