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Status |
Public on Mar 02, 2022 |
Title |
MaelKD_totalRNA_rep2 |
Sample type |
SRA |
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Source name |
BmN4 cells
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Organism |
Bombyx mori |
Characteristics |
cell type: Cultured cell derived from ovary cell line: BmN4 knockdown: siMael molecule subtype: total RNA rip antibody: none
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Treatment protocol |
To knockdown (KD) endogenous proteins, BmN4 cells (1 × 106 cells) were transfected with 500 pmol of siRNA duplex in 100 μL of EP buffer [137 mM NaCl, 5 mM KCl, 0.5 mM Na2HPO4 and 2.1 mM HEPES-KOH (pH 7.1)] using program T-001 of Nucleofector 2b device (Lonza). siRNA transfection was performed again to raise the RNAi effect. After KD, the cells were cultured for total 10 days and harvested. Luciferase (Luc) siRNA was used as a control. The sequences of siRNAs are summarized in Table S1. To exogenously express proteins, BmN4 cells (6 × 105 cells) were transfected with 2-8 μg of plasmids and 5-20 μL of FuGENE HD transfection reagent (Promega) in 100 μL of complete medium without FBS and added 10% FBS into cell medium 3 h later. After transfection, the cells were cultured for 2 days and harvested.
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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 10% fetal bovine serum (FBS) (Equitech-Bio) and penicillin-streptomycin-glutamine (Gibco).
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Extracted molecule |
polyA RNA |
Extraction protocol |
Total RNAs were isolated using ISOGEN II and DNase (Life technologies) and used to generate RNA libraries. RNA libraries were prepared from Luc, Mael, and Siwi KD BmN4 cells with VAHTS Stranded mRNA-seq Library Prep Kit for Illumina (Vazyme) and sequenced using Illumina NovaSeq to obtain paired-end reads.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
smRNA-seq: Analysis of PIWI-associated small RNA sequences were performed essentially as described previously (Iwasaki et al., 2017) with some modifications. After removal of adaptor sequences by cutadapt (version 3.1) (Martin et al., 2011), 22-35 nt reads were mapped to the genome using bowtie (version 1.3.0) (Langmead et al., 2009) in random and multiple counting methods not allowing mismatch. Genome mapped reads were processed using SAMtools (H. Li et al., 2009) and BEDTools (Quinlan and Hall, 2010) and used for further analysis with normalized by the number of the genome mapped reads. The genome mapped reads were aligned to the transposon library using bowtie in random and multiple counting methods allowing up to two mismatches. RNA-seq: Analysis of RNA sequences were performed essentially as described previously (Iwasaki et al., 2017) with some modifications. After cleaning and removal of adaptor sequences by using Rcorrector, TranscriptomeAssemblyTools, and TrimGalore, 36- nt reads were mapped to the genome and to the transposon library using STAR (version 2.7.9a) (Dobin et al., 2012) in random and multiple counting methods allowing up to 4% of mismatches relative to read length. Mapped reads were processed using SAMtools (Li et al., 2009) and BEDTools (Quinlan and Hall, 2010). Genome_build: silkworm reference genome obtained from Silkbase (Genome assembly; Nov. 2016) (Kawamoto et al., 2019) Supplementary_files_format_and_content: smRNA-seq: Tab-separated text files include normalized count of reads mapped to each transposon sequences in antisense direction. Supplementary_files_format_and_content: RNA-seq: Tab-separated text files include normalized count of reads mapped to each transposon sequences in sense direction.
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Submission date |
Jul 15, 2021 |
Last update date |
Mar 02, 2022 |
Contact name |
Yurika Namba |
E-mail(s) |
yurika-namba@g.ecc.u-tokyo.ac.jp
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Organization name |
The University of Tokyo
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Department |
Department of Biological Sciences, Graduate School of Science
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Lab |
Siomi Lab
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Street address |
2-11-16 Yayoi, Bunkyo-ku
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City |
Tokyo |
State/province |
--- Select a state --- |
ZIP/Postal code |
113-0032 |
Country |
Japan |
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Platform ID |
GPL29210 |
Series (1) |
GSE180191 |
Maelstrom functions in the production of Siwi-piRISC capable of regulating transposons in Bombyx germ cells |
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Relations |
BioSample |
SAMN20248538 |
SRA |
SRX11470421 |
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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