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| Status |
Public on May 04, 2018 |
| Title |
F12/gdv1-asKO 32-40 hpi |
| Sample type |
SRA |
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| Source name |
red blood cell stage, schizont
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| Organism |
Plasmodium falciparum |
| Characteristics |
developmental stage: red blood cell stage, schizont
strain: F12
genomic modification: gdv1-asRNA loss-of-function mutant line generated using CRISPR/Cas9-based genome editing
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| Treatment protocol |
F12 and F12/gdv1-asKO parasites were synchronized twice 16 hrs apart to obtain an eight hour growth window. Parasites were harvested at 32-40 hpi and 40-48 hpi.
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| Growth protocol |
P. falciparum F12 and F12/gdv1-asKO parasites were cultured as described (Trager et al. 1978). Growth synchronization was achieved by repeated sorbitol treatments (Lambros et al. 1979). F12/gdv1-asKO parasites were selected and cultured with 4 nM WR99210 (WR).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was isolated using Ribozol (Amresco) according to the manufacturer’s manual and further purified using the RNeasy Plus Mini Kit (Qiagen) for removal of gDNA. Residual gDNA was digested with TURBO DNA-freeTM DNaseI (Ambion) and the RNA was quantified by NanoDrop.
Isolated total RNA was PolyA-selected using the Oligotex mRNA Mini Kit (Qiagen, #70022) according to manufacturer’s instructions. Subsequently, 2µ of PolyA-selected total RNA equivalent were fragmented by alkaline hydrolysis (5x fragmentation buffer: 200mM Tris acetate pH 8.2, 500mM potassium acetate, 150mM magnesium acetate) for 1min 45sec at 85°C in a 150µl volume and precipitated as previously described in (Hoeijmakers, Bártfai and Stunnenberg 2013), followed by two TURBO DNase treatments (Ambion, #AM2238) to remove remnants of genomic DNA . Further library preparation for strand-specific RNA-seq was carried out as in (Kensche et al. 2015). In short, first strand cDNA synthesis was performed with AT-corrected Random N9 primers (76% AT) and in presence of 0.2µg Actinomycin D (Thermo 446 Fisher Scientific) to prevent unwanted DNA-dependent second strand cDNA synthesis. To maintain the strand-specific information during second strand synthesis dTTPs were replaced with dUTPs. Resulting double stranded cDNAs (1.9-3.2ng) were end repaired and extended with 3’ A-overhangs. After ligation of NextFlex adapters (Bio Scientific, #514122), libraries were treated with USER enzyme (NEB, #M5505L) to specifically degrade the dUTP-containing second strand and subsequently amplified by PCR (98°C for 2min; 4 cycles of 98°C for 20sec, 62°C for 3min; 62°C for 5 min) using KAPA HiFi HotStart ready mix (KAPA Biosystems, #KM2602) and NEXTflex primer mix (Bio Scientific, #514122). Next, libraries were size-selected for 300-400 bp fragments using 2% E-Gel Size Select agarose gels (Invitrogen, #G6610-02) and amplified for another 12 cycles using the same PCR protocol as above. Subsequent depletion of adapter dimers and library purification was performed using Agencourt AMPure XP beads (Beckman Coulter, #A63880) in a 1:1 library beads ratio.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
directional RNA-seq
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| 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) against the P.falciparum 3D7 transcriptome and reference genome (PlasmoDB version 26)
Sam files were converted to bam files and mapped reads were filtered to mapping quality ≥15 (SAM tools v1.2). Only uniquely mapped reads were used for further analysis.
Bam files were split into reads mapping to the sense strand (PlusStrand; FLAG16) and antisense strand (MinusStrand;FLAG0) respectively.
BedGraph files were generated to visualize directional RNA-seq data in the UCSC Genome Browser using bedtools genomecov with the option -bga and normalized using -scale and the scaling factor ‘1000000/amount of unique reads’.
Genome_build: P.falciparum 3D7 transcriptome and reference genome from PlasmoDB (version 26)
Supplementary_files_format_and_content: Bedgraph files - Purpose: Visualization of directional RNA-seq data in the UCSC Genome browser
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| Submission date |
Aug 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Sabine Fraschka |
| Organization name |
Radboud University Nijmegen
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| Department |
Molecular Biology
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| Lab |
Richárd Bártfai
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| Street address |
Geert Grooteplein 28
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| City |
Nijmegen |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
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| Platform ID |
GPL21298 |
| Series (1) |
| GSE94901 |
GDV1 triggers sexual conversion and differentiation in malaria parasites by antagonizing HP1-dependent gene silencing |
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| Relations |
| BioSample |
SAMN07511310 |
| SRA |
SRX3097552 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2743710_PfF12-GDV1asKO-40-48_MinusStrand.BedGraph.gz |
2.2 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM2743710_PfF12-GDV1asKO-40-48_PlusStrand.BedGraph.gz |
2.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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