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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 24, 2023 |
| Title |
Sap_mEx3_KD_BR1: N2a Flp-In rtTA (Sap30bp_withoutEx3), siSap30bp, Biological rep 1 |
| Sample type |
SRA |
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| Source name |
N2a Flp-In rtTA
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| Organism |
Mus musculus |
| Characteristics |
cell line: N2a Flp-In rtTA
cell type: Mouse neuroblastoma
cdna expressed_(n2a_flp-in_line): FLAG-Sap30bp_withoutExon3
condition: Sap30bp siRNA knockdown
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| Treatment protocol |
For siRNA knockdown and rescue experiments, N2a Flp-In rtTA cells (~1.2x105) were grown for 24 hours in two (Control) or three (Knockdown/Rescue) 6-well dishes and transfected with siGENOME siRNA (Dharmacon) using RNAiMax (Life Technologies) as recommended by the manufacturer. The siGENOME siRNA used for the transfections was Sap30bp (D-055094-01-0002) with a non-targeting control (NTC) pool (D-001810-10) used for the controls. Media was changed 24 hours post transfection with or without Doxycycline to induce isoform expression, with cells from all matching condition wells harvested and combined at 72 hours. Two-thirds of the total sample was stored for western blot analysis and one-third stored for RNA extraction.
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| Growth protocol |
Mouse neuroblastoma (N2a/N2a Flp-In rtTA) cells were grown in DMEM (high glucose; Sigma-Aldrich) supplemented with 10% FBS, sodium pyruvate, non-essential amino acids, and penicillin/streptomycin. Cell lines were maintained at 37°C with 5% CO2.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) as recommended by the manufacturer. For RT-PCR and RT-qPCR experiments, cDNA was synthesized for each sample for ~500ng - 1 µg of RNA using the Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Fischer Scientific, K1651) and diluted in water to a concentration of 5 ng/µL. qPCR reactions were performed using the SensiFAST SYBR No-ROX Kit (Meridian Bioscience, BIO-98005) and analyzed using a CFX96 Real-Time PCR Detection System (BIO-RAD).
1000 ng per sample was used in the library preparation. To obtain larger insert size (540 nt on average after size selection), Illumina’s TruSeq Stranded mRNA sample preparation protocol was modified as follows: 1) fragmentation time and temperature was reduced from 8 minutes at 98°C to 2 minutes at 80°C. Average size distribution prior to library preparation was 232 nt versus 740 nt, respectively; 2) after the final library was generated, the samples were loaded onto a 2% agarose gel (Cat # 1613101, BioRad) and run for 1 hour at 100 V. The libraries were size selected between 400 and 600 bp. Excised gel fragments were purified using the Qiagen MinElute Gel Extraction kit (Cat # 28604, Qiagen). The rest of the protocol was adapted as described in Illumina’s TruSeq Stranded mRNA sample preparation guide (Part# 15031047 Rev. E, Illumina).
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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 |
RTA 3.4.4
AS analysis for RNA-seq data was performed using the vast-tools analysis pipeline, version 2.2 https://github.com/vastgroup/vast-tools with VastDB version Mmu released Feb. 16, 2018 (Braunschweig et al., 2014; Irimia et al., 2014; Tapial et al., 2017). Events with poor coverage or junction balance were filtered out (vast-tools quality column score 3 other than SOK/OK/LOW for cassette exon [CE], microexon [MIC], and alternative 5' or 3' splice site [Alt5/3] events or coverage less than 15 reads for intron retention [IR] events; score 4 other than OK/B1 for CE and MIC events and score 5 of less than 0.05 for IR events). Differential splicing was assessed using a combination of the change in average “percent spliced in” (avgPSI) or percent intron retention (avgPIR) levels along with vast-tool’s diff module. For Sap30bp RNA-seq experiments, AS events showing changing splicing upon Sap30bp knockdown were defined as those with at least a 15% change in avgPSI/PIR (∆PSI/∆PIR) between the siRNA knockdown and NTC treated samples for both Sap30bp +Ex3 and ∆Ex3 expressing N2a Flp-In rtTA cell lines with maximum of 10% ∆PSI difference between cell lines and called as a significant change by the vast-tools diff module (minimum value for change with a likelihood of 0.95, MV[abs(dPSI)] at 0.95, > 0). Events showing Sap30bp mediated rescue by either the +Ex3 or ∆Ex3 isoforms were defined as those events showing significant Sap30bp knockdown in both cell lines (as described above) and for which the siRNA treated samples induced with Doxycycline showed at least a 10% avgPSI change compared to the siRNA knockdown samples (-Dox) and showed a significant change according to the vast-tools diff module (MV[abs(dPSI)] at 0.95 > 0). An event was called as showing isoform specific rescue if the change in the ∆PSI (∆∆PSI/∆∆PIR) between the rescue and knockdown samples was greater than 7% between the Sap30bp isoforms rescued cell lines. AS events showing Sap30bp regulation according to these criteria are listed in Supplementary table 5.
Gene expression changes were analyzed by pseudo-aligning pre-trimmed reads to GENCODE vM21 transcripts using Salmon v0.14.1 (Patro et al., 2017) and aggregated per gene using the R package tximport (Soneson et al., 2015). Differential expression was assessed using the classic mode (exactTest) in edgeR (McCarthy et al., 2012). Lowly expressed genes (<5 RPKM) were removed from the analysis. For Sap30bp knockdown, a gene was considered to show differential expression if it had at least a 0.6 log2FC (~50% change) between the siRNA knockdown and NTC control samples with an FDR < 0.1. For Sap30bp rescue samples, a gene was considered to be rescued by either Sap30bp isoform if it had at least a 0.4 log2FC between the Sap30bp rescue (+Dox) and siRNA knockdown samples in either +EX3 or ∆EX3 cell line with an FDR < 0.1.
Assembly: mm9
Supplementary files format and content: All files are in tab-delimited format. “vast.tools_INCLUSION_LEVELS_Altsplicing_Sap30bp_Mmu12-mm9.tab” contains vast-tools output showing the percent spliced in (PSI) values for each splicing events across samples. “AS.DiffEvents_Sap30bp_dPSI.15.tab” contains ∆PSI (dPSI) values and vast-tools diff module annotations for AS events showing differential inclusion between any of the NTC, Knockdown, or Rescue conditions (minimum ∆PSI of 15% with a likelihood of 0.95, MV[abs(dPSI)] at 0.95, > 0 [annotated as “TRUE” in signif. columns]). “DE_Sap30bp.tab” contains edgeR-estimated average RPKM, fold change and FDR values assessing differential RNA expression changes between conditions. (General Note: Sap.mEx.siNTC = ∆Exon3 isoform line_NTC, Sap.mEx.siSap = ∆Exon3 isoform line_Knockdown, Sap.mEx.siSap.dox = ∆Exon3 isoform line_Isoform Rescue, Sap.pEx.siNTC = +Exon3 isoform line_NTC, Sap.pEx.siSap = +Exon3 isoform line_Knockdown, Sap.pEx.siSap.dox = +Exon3 isoform line_Isoform Rescue)
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| Submission date |
Feb 09, 2023 |
| Last update date |
Nov 24, 2023 |
| Contact name |
Ulrich Braunschweig |
| Organization name |
University of Toronto
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| Department |
Donnelly Centre
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| Lab |
Benjamin J. Blencowe
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| Street address |
160 College Street
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5S 3E1 |
| Country |
Canada |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE224970 |
Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors (Sap30bp RNA-Seq) |
| GSE224971 |
Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors |
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| Relations |
| BioSample |
SAMN33229153 |
| SRA |
SRX19324664 |
| 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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