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| Status |
Public on Nov 24, 2023 |
| Title |
Total_Chtop_pEx5_KD_BR4: N2a Flp-In rtTA (Chtop_w/Exon5), siChtop, Total frac, Bio 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-Chtop_withExon5
fraction: Total
condition: Chtop 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 Chtop/2500003M10Rik (D-063383-02) 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 |
For nuclear/cytoplasmic fractionation experiments N2a Flp-In rtTA cells were grown for 72 hours in a 6-well dish as described above. After 72 hours, N2a cells were harvested through addition of 200 µL Trypsin (0.05%) for ~10 minutes until most cells could be dislodged from the plate with gentle shaking. After trypsinization, cells were resuspended in 500 µL of DMEM growth media and the total volume transferred to a 1.5 mL microcentrifuge tube. To ensure similar number of cells in each sample for lysis, cells were then counted using an automated cell counter (Millipore, Scepter Handheld Automated Cell Counter) and equal number of cells transferred to a new microcentrifuge tube corresponding to the lowest cell count within each experiment. Next, cells were pelleted by centrifugation (300 rcf for 3 minutes) and then washed two times with 500 µL PBS and samples placed on ice for all remaining steps. 20% (100 µL) of the sample was removed as a “total” sample, centrifuged (300 rcf for 3 mins at 4 °C), and cell pellets resuspended in 200 µL of pre-chilled RIPA buffer containing freshly added 1mM DTT and 1mM PMSF and stored on ice. The remaining sample (400 µL) was pelleted (300 rcf for 3 mins at 4 °C), and 200 µL pre-chilled RLN buffer (recipe from QIAGEN: “Purification of cytoplasmic RNA from animal cells using the RNeasy Mini Kit”) containing 50 mM Tris·Cl (pH 8.0), 140 mM NaCl, 1.5 mM MgCl2 and 0.5% (v/v) Nonidet P-40 with freshly added 1mM DTT and 1mM PMSF was added slowly, and mixed by carefully inverting the tube and gentle pipetting. Samples were left for 5 minutes until homogenous and then centrifuged (300 rcf for 3 mins at 4 °C) to pellet the nuclei and cell debris. The lysate (~200 µL total) was removed and centrifuged at maximum speed (17,000 rcf for 3 mins at 4 °C) to remove any undigested cell debris and lysate transferred to a new tube (the “cytoplasmic” fraction). The remaining pellet (containing nuclei) were gently washed again with pre-chilled 200 µL RLN buffer as described above and pelleted again by centrifugation (300 rcf for 3 minutes at 4 °C). Pellets were then resuspended in 200 µL of RIPA buffer (the “nuclear” fraction). All samples were sonicated (2x15 pulses) to help sample homogenization before the lysates were divided with approximately one-third of the sample (~80 µL) taken for protein analysis and approximately two-thirds (~120 µL) taken for RNA analysis. For protein samples, Laemmli buffer was added and samples stored at -80 °C until western blots performed as described above. RNA was purified directly (same day) from the RLN buffer lysates using the RNeasy Mini Kit (QIAGEN) as recommended by the manufacturer and samples prepared for sequencing.
RNA samples (250 ng) were enriched for mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490L, NEB) and subsequent libraries obtained using the NEBNext Ultra II Directional RNA library prep kit (E7760L, NEB) with some modifications. Specific modifications include; 1) fragmentation time was reduced from 15 minutes at 94°C to 7.5 minutes, 2) During the first strand cDNA synthesis reaction, the incubation times were increased from from 15 to 50 minutes at 42°C and 3) fragments were size selected using Ampure XP Beads (17.5ul for removing large fragments and 10ul to retain fragments without dimers).
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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
Pre-trimmed reads from total, nuclear, and cytoplasmic samples were pseudo-aligned to GENCODE vM21 transcripts using Salmon v0.14.1 (Patro et al., 2017) and gene-level abundances calculated using tximport (Soneson et al., 2015). Differential analysis comparing the nuclear/cytoplasmic ratios in the Chtop knockdown and rescue fractions was performed using DESeq2 (Love et al., 2014) as per Lee et al., 2020 with a minimum of 3 RPKM required across all samples. Gene-level changes in the knockdown vs control and rescue vs knockdown samples were calculated by comparing nuclear/cytoplasmic ratios across samples using the formula log2(Nuclear/Cytoplasmic)kd/resc – log2(Nuclear/Cytoplasmic)ntc/kd). Changes in nuclear (formula: log2(Nuclear/Total)kd/resc - log2(Nuclear/Total)ntc/kd) and cytoplasmic (formula: log2(Cytoplasmic/Total)kd/resc– log2(Cytoplasmic/Total)ntc/kd) fractions were similarly analyzed using DESeq2 normalized by the totals. For knockdown (kd vs ntc) comparisons, a transcript was considered to show an altered nuclear/cytoplasmic ratio in the siRNA knockdown samples compared to controls if the change in nuclear/cytoplasmic ratios was greater than 0.3 (~20% change) with a similar change (>0.3) in either the nuclear or cytoplasmic fractions. For Chtop rescue (resc vs kd) samples, a transcript was considered to be rescued by either Chtop isoform (Chtop +Ex5 or Chtop ∆Ex5) if it showed a change in the knockdown samples (as described above) and had at least a 0.25 change in nuclear/cytoplasmic ratio between the Chtop rescue (+Dox) and matching siRNA knockdown samples from the same expression line in the opposite direction as the knockdown. A transcript was annotated as showing better rescue upon re-expression of either Chtop isoform if the difference in the change in nuclear/cytoplasmic ratios between the Chtop isoform expressing lines was greater than 0.25 (∆∆log2(Nuclear/Cytoplasm) ratio).
Assembly: mm10 (GENCODE vM21)
Supplementary files format and content: All files are in tab-delimited format. “NCFracts_Chtop_COUNTS.txt.gz” and “NCFracts_Chtop_RPKM.tab.gz” contains the raw count and RPKM values for genes expressed (3 RPKM) across all conditions (NTC/Kd/Resc) and fractions (Total/Nuclear/Cyto). “NCFracts_Chtop_DESeq2_normCOUNTS_CytovsTotal_wAnnotations.txt.gz”, “NCFracts_Chtop_DESeq2_normCOUNTS_NucvsCyto_wAnnotations.txt.gz” and “NCFracts_Chtop_DESeq2_normCOUNTS_NucvsTotal_wAnnotations.txt.gz” contains the normalized count information as calculated by DESeq2 along with the Nuclear/Cytoplasmic, Nuclear/Total and Cytoplasmic/Total log2FC values used to assess changes in RNA expression between the fractions. Genes showing changing ratios in the knockdown (KD vs NTC) or isoform rescue (RES vs KD; mEX5/pEX5) samples are highlighted in the “Annotation” columns (“BOTH” = consistent regulation with both isoform rescue samples [>0.25 ∆log2FC and <0.25 ∆∆log2FC_pEXvsmEX], mEX_Up/Dn = increased or decreased [>0.25 ∆log2FC and >0.25 ∆∆log2FC_pEXvsmEX] nuclear/cytoplasmic expression with the ∆Ex5 isoform, pEX_Up/Dn = increased or decreased [>0.25 ∆log2FC and >0.25 ∆∆log2FC_pEXvsmEX] nuclear/cytoplasmic expression with the +Ex5 isoform).
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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) |
| GSE224969 |
Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors (Chtop RNA-Seq) |
| GSE224971 |
Analysis of alternative exon-dependent interactome remodelling reveals multitasking functions of gene regulatory factors |
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| Relations |
| BioSample |
SAMN33229135 |
| SRA |
SRX19323822 |
| 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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