Status
Public on May 22, 2025
Title
RNAseq_ESC_ContsiRNA_rep1
Sample type
SRA
Source name
E14
Characteristics
cell line: E14
cell type: Mouse embryonic stem cells (ESCs)
genotype: WT
treatment: ESCs were transfected with control siRNA and collected 3 days post transfection
Treatment protocol
8x10^4 parental ESCs were seeded into gelatin-coated 6 cm plate. Cells were let to attach for 4 hour and then transfected with 2 μl of 40 μM siRNA stock using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, 13778150) following the manufacturing instructions
Growth protocol
Mouse embryonic stem cells (E14) were cultured in N2B27 medium with LIF/2i
Extracted molecule
total RNA
Extraction protocol
Total RNA was purified with TRIzol reagent (Life Technologies).
The quality of the isolated RNA was determined with a Qubit® 4 Fluorometer and 4200 Tape Station system. Only those samples with a 260nm/280 nm ratio between 1.8–2.1 and a 28S/18S ratio within 1.5-2 were further processed. The TruSeq Stranded mRNA (Illumina) was used in the succeeding steps. Briefly, total RNA samples (100-1000 ng) were polyA enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired and adenylated before ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit® 4 Fluorometer. The product is a smear with an average fragment size of approximately 260 bp. Libraries were normalized to 10 nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. TheIllumina NovaSeq X Pluswas used for cluster generation and sequencing according to standard protocol. Sequencing was paired end at 2x150 bp.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
cDNA
Instrument model
Illumina NovaSeq X Plus
Description
RNAseq_sample_1
Data processing
The quality of the 150 bp paired end reads generated by the machine was checked by FastQC (Andrews, 2010). The quality of the reads was increased by applying: a) SortMeRNA (Kopylova, 2012) (version 2.1) tool to filter out ribosomal RNA; b) Trimmomatic (Bolger, 2014) (version 0.36) software package to trim the sorted (a) reads. The sorted (a), trimmed (b) reads were mapped against the mouse genome (mm10) using the default parameters of the STAR (Spliced Transcripts Alignment to a Reference, version 2.4.0.1) (Dobin et al., 2013). For each gene, exon coverage was calculated using a custom pipeline and then normalized in reads per kilobase per million (RPKM) (Mortazavi et al., 2008), the method of quantifying gene expression from RNA sequencing data by normalizing for total read length and the number of sequencing reads.
Assembly: mm10 (GRCm38)
Supplementary files format and content: xlsx
Supplementary files format and content: Excel file includes the DESeq2 computed mean of normalized count of all samples based on sequencing depth
Submission date
Jun 24, 2024
Last update date
May 22, 2025
Contact name
Raffaella Santoro
Phone
+41 44 635 54 75
Organization name
University of Zurich
Department
Dep. of Molecular Mechanisms of Disease
Street address
Winterthurerstrasse 190
City
Zurich
ZIP/Postal code
8057
Country
Switzerland
Series (2)
GSE270573
The nucleolar granular component is required for NAD association with nucleoli and the establishment of repressive chromatin architecture [RNAseq_EHMT2KD]
GSE270780
The nucleolar granular component mediates genome-nucleolus interactions and establishes their repressive chromatin states
Relations
Supplementary data files not provided
Raw data are available in SRA