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| Status |
Public on Sep 17, 2024 |
| Title |
membrane, 120min, replicate2 |
| Sample type |
SRA |
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| Source name |
E14TG2a
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| Organism |
Mus musculus |
| Characteristics |
fraction: membrane
time point: 120min
cell line: E14TG2a
cell type: mESC
treatment: 1microM flavopiridol
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| Treatment protocol |
Three independently passaged biological replicates of mESCs (~3.5 × 107 cells per replicate) were cultured in “2i + LIF” ES medium supplemented with 1µM flavopiridol to block the transcription.
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| Growth protocol |
E14TG2a mESCs [Iacovino, M., Roth, M. E. & Kyba, M. Rapid genetic modification of mouse embryonic stem cells by Inducible Cassette Exchange recombination. Methods Mol. Biol. 1101, 339–351 (2014)] were cultured in 0.1% gelatin [w/v] coated plates in “2i + LIF” ES medium [Advanced DMEM/F12 (12634028, Thermo Fisher Scientific) – Neurobasal (21103049, Thermo Fisher Scientific) – Knockout™ DMEM (10829018, Thermo Fisher Scientific) (1 : 1 : 0.5), 14% Fetal Bovine Serum qualified for ES cells (16141079, Life Technologies), 1X N2 (17502048, Thermo Fisher Scientific), 1X B27 (17504001, Thermo Fisher Scientific), 1X GlutaMax (35050061, ThermoFisher Scientific), 1X MEM Non-Essential Amino Acid (11140050, Thermo Fisher Scientific), 1X Nucleosides (ES-008-D, Merck Millipore), 100 µM β-mercaptoethanol (21985023, Thermo Fisher Scientific), 3 μM CHIR99021 (SML1046-5MG, Invitrogen) and 1 μM PD0325901 (PZ0162-5MG, Invitrogen), 1000 U/ml Leukemia inhibitory factor (ESG1107, Merck Millipore)], under a controlled atmosphere at 5% CO2 and 37°C. mESCs were seeded the day before the experiments at a density of 3× 105 cells/ml.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells were harvested at 0, 30, 60, 120 and 180 min after addition of flavopiridol followed by cell fractionation. Briefly, medium was aspirated, 5 ml of ice-cold PBS supplemented with 100 µM cycloheximide (A0879,0001, Biochemika) was added to the plates, cells were scraped from the plates, transferred to 15ml falcon and spun down. Pellet was resuspended in 500 µl of ice-cold permeabilization buffer (110 mM KOAc, 25 mM K-HEPES pH 7.2, 2.5 mM Mg(OAc)2, 1 mM EGTA with freshly added 0.015% digitonin, 1 mM DTT, 100 μg/ml cycloheximide, 1X Complete Protease Inhibitor Cocktail and 40 U/mL RNaseOUT™). 100 µl of the sample was taken aside as Total extract and rest was incubated for 10 min at 4°C with rotation, followed by centrifugation at 3000 g 5 min at 4°C. Supernatant (corresponding to the Cytosolic fraction) was transferred to new tube while the pellet was resuspended in 5ml of wash buffer (110 mM KOAc, 25 mM K-HEPES pH 7.2, 2.5 mM Mg(OAc)2, 1 mM EGTA with freshly added 0.004% digitonin, 1mM DTT, 100μg/ml cycloheximide) and spun down again at 3000 g 5 min at 4°C. After centrifugation washed pellet was mixed with 500 µl of ice-cold lysis buffer (400 mM KOAc, 25 mM K-HEPES pH 7.2, 15 mM Mg(OAc)2, 0.5% (v/v) NP-40 and freshly added 1 mM DTT, 100 μg/ml cycloheximide, 1X Complete Protease Inhibitor Cocktail, 40 U/mL RNase Out) and incubated for 5 min on ice followed by centrifugation at 3000 g 5 min at 4°C to collect the supernatant (corresponding to the Membrane fraction) and the pellet (insoluble and the nuclear fraction). For additional purity nuclei were loaded on 10% sucrose cushion in lysis buffer and centrifuged at 200 g 5 min at 4°C. The cytosolic and membrane fractions were clarified at 7500 g 10 min at 4°C to remove cell debris.Obtained fractions were mixed with Trizol LS and RNA was isolated following the manufacturer instructions.
One microgram of total RNA was used as an input for TruSeq Stranded mRNA Library Prep Kit according to the instructions of the manufacturer.
TruSeq Stranded mRNA Library Paired-End
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Reads were aligned with STAR (v2.7.6) and counted with RSEM (v1.3.3).
Library sizes were normalized using genes with known half-lives greater than 14 hours (Herzog et al, 2017). Samples of each time series were further normalized to the corresponding t=0 time point.
We fitted a simple exponential decay model per compartment for roughly 10,000 genes, giving the aggregated subcellular turnover rates, in contrast to the rates of transition from one compartment to the next.
Assembly: mm10
Supplementary files format and content: {sample}_norm_read_count.txt files provide normalized read counts per gene.
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| Submission date |
Feb 22, 2024 |
| Last update date |
Sep 17, 2024 |
| Contact name |
Markus Landthaler |
| E-mail(s) |
markus.landthaler@mdc-berlin.de
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| Organization name |
Max Delbrueck Center
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| Department |
Berlin Institute for Medical Systems Biology
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| Street address |
Hannoversche Str. 28
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| City |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE256335 |
Quantification of subcellular mRNA kinetics in mouse embryonic stem cells (flavopiridol) |
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| Relations |
| BioSample |
SAMN40033415 |
| SRA |
SRX23698313 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8093929_m120min2_norm_read_count.txt.gz |
177.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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