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Sample GSM5455099

Query DataSets for GSM5455099

Status

Public on Mar 02, 2022

Title

MaelKD_Siwi-piRNA_rep2

Sample type

SRA

Source name

BmN4 cells

Organism

Bombyx mori

Characteristics

cell type: Cultured cell derived from ovary
cell line: BmN4
knockdown: siMael
molecule subtype: small RNA
rip antibody: anti-FLAG

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.

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).

Extracted molecule

total RNA

Extraction protocol

Dynabeads Protein G (Invitrogen) and anti-Flag antibodies (Sigma, M2 mouse monoclonal F3165) were incubated in T-PBS containing 0.02% Tween at room temperature. BmN4 cells were lysed at 4°C with immunoprecipitation (IP) buffer [30 mM HEPES-KOH (pH 7.3), 150 mM KOAc, 2 mM Mg(OAc)2, 5 mM DTT, 0.1% NP-40, 2 μg/mL pepstatin, 2 μg/mL leupeptin, and 0.5% aprotinin] containing RNasin Plus Ribonuclease Inhibitor (Promega). After the cell debris was removed by centrifugation, the cell lysates were incubated with antibodies-conjugated beads for 1 h at 4°C. After protein-antibody binding, the beads were washed once with IP buffer containing 500 mM NaCl and then extensively with IP buffer. The protein-bound RNAs were purified from the beads by proteinase K (Roche) treatment, phenol/CHCl3 treatment, and precipitated with ethanol. RNAs of 22–35 nt in length were separated on denaturing (6 M urea) PAGE and used to generate RNA libraries.
PIWI-associated small RNA libraries were prepared from Luc, Mael, and Ago3 KD BmN4 cells with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) and sequenced using Illumina MiSeq to obtain 51 nt single reads.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina MiSeq

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.

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

Organization name

The University of Tokyo

Department

Department of Biological Sciences, Graduate School of Science