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| Status |
Public on Jun 30, 2018 |
| Title |
6h_R-_3 |
| Sample type |
SRA |
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| Source name |
inducible AP2-G overexpression line
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| Organism |
Plasmodium berghei |
| Characteristics |
tissue: Plasmodium berghei cells
host: Female, outbred Theiler's Original (TO) mice
rapamycin treated?: NO
time (hours post induction): 6h
plasmodium strain: inducible AP2-G overexpression line
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| Growth protocol |
Female, outbred Theiler’s Original (TO) mice, 8-12 weeks old were injected with PBGAMi parasites synchronized to late schizont stage. The gametocyte conversion was induced in half of the animals by intraperitoneal injection of 4 mg/kg of rapamycin. Blood containing the developing parasites was harvested at different time point post induction (0h, 6h, 12h, 18h, 24h and 30h) via cardiac puncture. Leucocyte depletion was achieved by filtration of the blood through two sequential Plasmodipur ® filters (Europroxima) according to the manufacturer’s instructions. Remaining red blood cells were resuspended in 10 volumes of pre-chilled erythrocyte lysis buffer (150mM NH4Cl; 10mM KHCO3; 1mM EDTA) and incubated on ice for 15min. After lysis parasites were pelleted by centrifugation for 8 minutes at 450g and washed with 1x phosphate buffered saline until complete removal of the coloration of the supernatant. Parasite pallet was resuspended in 1ml of Trizol reagent (Ambion) lysed for 10 min at room temperature and stored at -80°C for later RNA extraction. For each time point at least two independent biological replicates of treated parasites and matched untreated controls were generated.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Complete RNA was isolated from the samples using Trizol/chloroform extraction followed by isopropanol precipitation and its concentration and integrity was verified using Agilent Bioanalyzer (RNA 6000 Nano kit) and NanoDrop 1000 spectrophotometer.
1-2 µg of total RNA from each sample (all complete sample if the yield was lower) was used for mRNA isolation (Magnetic mRNA Isolation Kit, NEB). First strand cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System and a 1:1 mix of Oligo(dT) and random primers (Invitrogen). The DNA/RNA hybrids were purified using Agencourt RNACleanXP beads (Beckman Coulter) and the second cDNA strand was synthesized using a 10 mM dUTP nucleotide mix, DNA Polymerase I (Invitrogen) and RNAseH (NEB) for 2.5 h at 16°C. The long cDNA fragments were purified and fragmented using a Covaris S220 system (duty cycles= 20, intensity = 5,cycles/burst =200,time= 30s). The ~200 bp long fragments were end-repaired, dA-tailed and ligated to “PCR-free” adapters (Kozarewa et al., 2009) with index tags using NEBNext kits according to the manufacturer’s instructions. Excess adapters were removed by two rounds of clean-up with 1xvolume of Agencourt AMPure XP beads. Final libraries were eluted in 30μl water, quality-controlled using Agilent Bioanalyzer (High Sensitivity DNA chip) digested with USER enzyme (NEB) and quantified by qPCR. For some libraries, additional 5 cycles of PCR amplification were performed, using KAPA HiFi HotStart PCR mix and tag-specific primers to obtain enough material for sequencing. Pools of indexed libraries were sequenced using an Illumina HiSeq2500 system (100 bp paired-end reads) according to manufacturer’s manual.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
base calling, quality checking, index sorting and generation of initial *cram files performed using the automatic pipelines of the sequencing service of the Wellcome Trust Sanger Institute. All details of the processing are included in the *cram file header.
concatenation of the *cram files from the two lanes of HiSeq2500 run using samtools (v.1.3.1)
generation of *fastq file from *cram files using Picard-tools SamToFastq (v.2.3)
re-alignment of the reads with from *fastq files against the reference genome in a splice-aware manner using hisat2-2.0.0-beta software and generation of *sam files
sorting and indexing of the resulting *sam files using samtools (v.1.3.1)
counting of the reads aligned to each of the Plasmodium genes using htseq-count command from HTSeq Python library (v. 0.6.1) with option '-s reverse' and gff file matching the reference genome, http://plasmodb.org/common/downloads/release-30/PbergheiANKA/gff/data/
Genome_build: http://plasmodb.org/common/downloads/release-30/PbergheiANKA/fasta/data/
Supplementary_files_format_and_content: *.htseq_count files containing the raw read counts for all the genes in Plasmodium berghei genome
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| Submission date |
Feb 06, 2018 |
| Last update date |
Jun 30, 2018 |
| Contact name |
Katarzyna Kinga Modrzynska |
| E-mail(s) |
[email protected]
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| Phone |
+44 141 330 4599
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| Organization name |
Wellcome Trust Centre for Molecular Parasitology
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| Street address |
B627 Sir Graeme Davies Building, 120 University Place
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| City |
Glasgow |
| State/province |
Glasgow (City of) |
| ZIP/Postal code |
G12 8TA |
| Country |
United Kingdom |
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| Platform ID |
GPL20293 |
| Series (1) |
| GSE110201 |
Transcriptome changes during inducible gametocytogenesis in Plasmodium berghei parasites |
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| Relations |
| BioSample |
SAMN08470886 |
| SRA |
SRX3653212 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2982196_21384_6.htseq_count.txt.gz |
23.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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