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Sample GSM5455105

Query DataSets for GSM5455105

Status

Public on Mar 02, 2022

Title

MaelKD_totalRNA_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: total RNA
rip antibody: none

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

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

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