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Sample GSM7133770

Query DataSets for GSM7133770

Status

Public on May 28, 2024

Title

N2A cells, Vorinostat_SAHA, replicate A

Sample type

SRA

Source name

N2A

Organism

Mus musculus

Characteristics

cell line: N2A
cell type: Mouse neuroblastoma cells
treatment: Vorinostat_SAHA

Treatment protocol

N2A cells were seeded into 6-well plates. The following day, cells were treated with small molecules for a period of 24 hours (0.4% DMSO final concentration).

Growth protocol

Mouse Neuro-2A (N2A) cells were grown in DMEM supplemented with 10% FBS, sodium pyruvate, MEM non-essential amino acids, and penicillin/streptomycin, and maintained at 37°C with 5% CO2.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA purification was performed using RNeasy spin columns (Qiagen) following manufacturer's instructions. An on-column DnaseI treatment was performed using RNase-free DNase set (Qiagen) following manufacturer's instructions.
Illumina TruSeq RNA Library Prep Kit v2

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

C02.Vorinostat_SAHA.A

Data processing

Base-calling Q-scores were obtained with RTA 3.4.4
For alternative splicing analysis, RNA-Seq data were processed using the vast-tools pipeline, version 2.1 https://github.com/vastgroup/vast-tools (Braunschweig et al., 2014; Irimia et al., 2014; Tapial et al., 2017). Events were filtered for coverage and junction balance, requiring a vast-tools quality column score 3 of SOK/OK/LOW for cassette exon (CE), microexon (MIC), and alternative 5' or 3' splice site (Alt5/3) events or at least 15 reads for intron retention (IR) events and a balance score (score 4) of ‘OK’ or ‘B1’ for alternative exons or > 0.05 for intron retention events, in at least half of replicate experiments. If more than one replicates was present, the point estimate from vast-tools diff was used and if the coverage/balance filtering step failed for an event in more than half of the replicates, its value was set to NA. Changes were considered significant if they were greater than 10 ∆PSI/dPIR and the expected minimum change was different from zero at p > 0.95 according to vast-tools’ diff module.
Gene expression changes were analyzed by pseudo-aligning pre-trimmed reads to GENCODE vM17 transcripts using Salmon v0.10.2 (Patro et al., 2017) and aggregated per gene using the R package tximport (Soneson et al., 2015). Differential expression was then assessed using the classic mode (exactTest) edgeR (McCarthy et al., 2012), and genes with an FDR < 0.05 and a greater than 2-fold change were considered differential. For each comparison, only genes expressed at a minimum of 5 RPKM in one or both conditions were considered.
Assembly: mm9
Supplementary files format and content: vast-tools.AltSplicing.tab.gz is a tab-delimited file and contains vast-tools output, i.e. event information and raw percent-spliced-in values and quality scores per sample. See https://github.com/vastgroup/vast-tools#combine-output-format.
Supplementary files format and content: AltSplicing.tab.gz is a tab-delimited file and contains mean percent-splice-in values per treatment and significance calls for the difference vs. DMSO control.
Supplementary files format and content: Expression.tab.gz is a tab-delimited file and contains RPKM, log2 fold-change and p-values per treatment vs, DMSO control.

Submission date

Mar 30, 2023

Last update date

Aug 11, 2024

Contact name

Ulrich Braunschweig

Organization name

University of Toronto

Department

Donnelly Centre

Lab

Benjamin J. Blencowe