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| Status |
Public on Jan 25, 2022 |
| Title |
WT_PAPI-CLIP-seq_rep3 |
| Sample type |
SRA |
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| Source name |
BmN4 cells
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| Organism |
Bombyx mori |
| Characteristics |
genotype: WT
cell type: BmN4
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| Treatment protocol |
BmN4 cells were transfected with 2 μg of plasmids (per 6 × 105 cells) using FuGENE HD (Promega). After 48 h, transfected cells were harvested for FAST-iCLIP analysis.
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| Growth protocol |
BmN4 cells were cultured at 26 °C in EX-CELL® 420 Serum-Free Medium for insect cells (Sigma) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio) and penicillin-streptomycin-glutamine (Thermo Fisher Scientific).
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| Extracted molecule |
total RNA |
| Extraction protocol |
10 cm dish BmN4 cells transfected Papi-Flag or Papi1-222-Flag were used for each iCLIP experiment. For UV crosslinking, BmN4 cells were washed once with ice-cold PBS. The UV crosslinking was performed with irradiance of 200 mJ/cm2 at 254 nm. Cells were pelleted and lysed with lysis buffer [20 mM HEPES (pH7.3), 150 mM sodium chloride, 1 mM EDTA, 1 mM DTT, 0.5% Triton X-100, 2 μg/mL pepstatin, 2 μg/mL leupeptin, 0.5% aprotinin]. RNA–protein complexes were immunoprecipitated using 12 μg of anti-FLAG M2 antibody and 120 μL of Dynabeads Protein G at 4 °C for 2 h. Beads were washed three times with wash buffer [20 mM HEPES (pH7.3), 300 mM sodium chloride, 1 mM DTT, 0.5% Triton X-100, 2 μg/mL pepstatin, 2 μg/mL leupeptin, 0.5% aprotinin], followed by partial RNA digestion with 1 U/μL RNase T1 for 15 min at room temperature. Beads were washed three times with high-salt wash buffer [20 mM HEPES (pH7.3), 500 mM sodium chloride, 1 mM DTT, 0.5% Triton X-100, 2 μg/mL pepstatin, 2 μg/mL leupeptin, 0.5% aprotinin]. After 3' end RNA dephosphorylation with T4 PNK, the RNAs were ligated at their 3' end s to a preadenylated RNA adaptor with T4 RNA ligase2, truncated KQ (New England Biolabs), and then radioactively labeled. The samples were analyzed by NuPAGE gel electrophoresis (Life Technologies) and RNA–protein complexes were transferred to nitrocellulose. After cutting out the region of nitrocellulose containing RNA–protein complexes, RNAs were removed from the membrane by Proteinase K (Roche) digestion.
Extracted samples were processed for reverse transcription, cDNA purification, and cDNA circularization as previously described (Flynn et al., 2015). Libraries were then amplified with Q5 DNA polymerase (New England Biolabs) and size-selected via polyacrylamide gel electrophoresis.
iCLIP libraries were sequenced using the MiSeq platform as single-end 51-bp reads.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Reads were trimmed by a 3′ adapter sequence using Cutadapt (Martin, 2011), and those shorter than 27bp were discarded. PCR duplicates were removed by collapsing all identical reads containing the same random barcode using fastx-collapser from FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit), followed by the removal of 5′-end random barcode at positions 1–9 of the reads. Mapping was performed against the Bombyx mori genome assembly (Nov.2016) from the SilkBase database (Kawamoto et al., 2019) using STAR (Dobin et al., 2013).
Genome_build: Bombyx mori genome assembly (Nov.2016) from the SilkBase database (Kawamoto et al., 2019)
Supplementary_files_format_and_content: 3′ adapter sequence trimmed, size selected, PCR duplicates removed, and barcodes are removed. Then reads were mapped to the silkworm genome, and mapped reads were extracted. For each unique CLIP tag sequences, read counts are indicated in tab delimited format.
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| Submission date |
Jul 13, 2021 |
| Last update date |
Jan 25, 2022 |
| Contact name |
Yuka W. Iwasaki |
| E-mail(s) |
iwasaki@keio.jp
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| Organization name |
Keio University School of Medicine
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| Department |
Department of Molecular Biology
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| Lab |
Siomi Lab
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| Street address |
35 Shinanomachi, Shinjuku-ku,
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| City |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
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| Platform ID |
GPL18754 |
| Series (1) |
| GSE179432 |
Siwi cooperates with Par-1 kinase to regulate mitochondrial Papi scaffolding Siwi-piRISC biogenesis |
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| Relations |
| BioSample |
SAMN20195704 |
| SRA |
SRX11426134 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5444688_Papi-F-WT-3_CLIPtags.txt.gz |
996.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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