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| Status |
Public on Oct 17, 2019 |
| Title |
PfDis3_R_rep2_RIP-seq |
| Sample type |
SRA |
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| Source name |
Pfdis3 RIP
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| Organism |
Plasmodium falciparum |
| Characteristics |
development stage: Ring
host: Homo sapiens
rip antibody: mouse anti-Ty1,Sigma SAB4800032-50 ug
genotype: wildtype
strain: Pf3D7
rip: Pfdis3 RIP
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| Extracted molecule |
total RNA |
| Extraction protocol |
Infected red blood cells (iRBCs) were collected by centrifugation and resuspended in cold PBS(10 volume of iRBC) . Parasites were extracted with 0.15% saponin and washed with cold PBS until the supernatant was clear. Lysis buffer(50 mM Tris-Cl pH7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100/ NP-40)with protease inhibitors and RNase inhibitors was added to the parasite pellet[1.5ml/ (1×109 parasites)] and incubated with rotation for 1h at 4℃. Cell debris were spinned out at 13000rpm for 15min at 4℃ and supernatant was incubated with 10ug of anti-Ty1 antibodies at 4℃ for 3h with rotation. For preparation of equilibrated protein-G magnetic beads, 50 ul protein-G magnetic beads were washed once with Wash Buffer(10mM Tris-Cl pH 7.5, 150mM MgCl2, 150mM KCl, 0.1% Triton X-100), then washed once with Elution Buffer, and washed twice with Wash Buffer. Protein-G magnetic beads were mixed with supernatant with protease inhibitors and RNase inhibitors at 4℃ overnight with rotation. Beads were washed twice with 500 ul Wash Buffer and once with PBS and then resuspended in 700ul TRIZOL at 4℃ for 10 min. After removed the beads, 140ul chloroform was added to trizol at room temperature for 5 min and mixture was centrifuged at 13800rpm for 30 min at 4℃. Supernatant was added to a new 1.5ml centrifuge tube and centrifuged at 13800rpm for 2 min at 4℃. Supernatant was mixed with 1ul glycogene and equal volume of isopropanol on ice for 2h. Tube was centrifuged at 13800rpm for 30 min at 4℃. Pellet was washed twice with 1 ml of 75% (vol/vol) ethanol, then air-dried and resuspended in 20µl of RNase-free water[3]. DNase I was used to digest DNA for 15min at room temperature. RNase-free water was added up to 100μl, mixed with equal volume of chloroform/isoamyl alcohol pH<5.0 (24:1), and then mixture was centrifuged at 13500rpm for 5 min. Supernatant was mixed with phenol/chloroform/isoamyl alcohol (24:1), and centrifuged at 13500rpm for 5 min. Supernatant was transferred to a new 1.5ml centrifuge tube with 1ul glycogene, one-tenth volumes of 3 M sodium acetate(pH 5.2), and 2.5 volumes of 100% ethanol and incubated at −80 °C for 20min. Samples were centrifuged at 13500rpm for 15 min and pellet was washed twice with 1 ml of 75% (vol/vol) ethanol, then air-dried and resuspended in 11µl of RNase-free water.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Low-quality and adaptor sequences were trimmed from the reads using cutadapt (v1.16) with parameters: -a AGATCGGAAGAGC -A AGATCGGAAGAGC --trim-n -m 50 -q 20,20.
Then, the reads were mapped to the Plasmodium falciparum 3D7 genome (Pf 3D7 v32, obtained from PlasmoDB) using Hisat2 strand-specific mode (v2.1.0) with parameters: --rna-strandness RF --dta --no-discordant --no-mixed --no-unal.
The Samtools (v1.9) was used to transfer the mapping results from sam format to position sorted bam format.
Mapped reads were subsequently assembled into transcripts guided by the PlasmoDB gff annotation files (Pf 3D7 v32) using featureCounts (v1.6.1) with parameters: -M -p -B -C for all; -s 2 for sense transcripts; -s 1 for antisense transcripts.
Genome_build: Pf 3D7 v32
Supplementary_files_format_and_content: The raw counts were calculated by featureCounts as described above.
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| Submission date |
Jul 01, 2019 |
| Last update date |
Oct 17, 2019 |
| Contact name |
meng liu |
| E-mail(s) |
1710949@tongji.edu.cn
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| Organization name |
TongJi University
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| Street address |
1239,SiPing Road
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| City |
shanghai |
| ZIP/Postal code |
20082 |
| Country |
China |
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| Platform ID |
GPL26835 |
| Series (2) |
| GSE133652 |
TRIBE uncovers the role of Dis3 in shaping the dynamic transcriptome in malaria parasites [RIP-seq] |
| GSE133654 |
TRIBE uncovers the role of Dis3 in shaping the dynamic transcriptome in malaria parasites |
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| Relations |
| BioSample |
SAMN12175153 |
| SRA |
SRX6386823 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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