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| Status |
Public on Sep 17, 2024 |
| Title |
cytosol, 0min, rep3 |
| Sample type |
SRA |
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| Source name |
E14TG2a
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14TG2a
cell type: mESC
treatment: 500uM 4-thiouridine
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| Treatment protocol |
Two independently passaged biological replicates of mESCs (~3.5 × 107 cells per replicate) were separately labelled in “2i + LIF” ES medium supplemented with 500 µM (for 0, 15, 20, 30, 40 min) or 100 µM (for 60, 120 and 180 min) 4-thiouridine (4sU, RP-2304, ChemGenes) and fractionated by sequential detergent extraction, as described previously [Jagannathan et al. 2011] with minor modifications.
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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 |
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. 20 µl of all fractions were taken for Western analysis while rest were mixed with Trizol LS (10296028, Thermo Fisher Scientific) for subsequent RNA isolation.
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 |
Alignment of raw reads was done with STAR (v2.7.6).
Reads in exons were counted with HTSeq-count (v0.11.1) and exon and TPM values were obtained by RSEM (v.1.3.3) using default parameters.
Based on a binomial mixture model by Juerges et al. (2018) we determined the conversion rate per sample. With the conversion rates, T and T2C conversion counts the fraction of new to total RNA was calculated on intron/exon level, which was used later on as input for the kinetic model.
Gene set enrichment analyses were performed with fgsea (v1.28.0) with the GSEA parameter set to 0 and gene set sizes limited from 30 to 900.
Fitting was performed throughout with the python package lmfit (v1.0.3) using the Levenberg-Marquardt “least-squares” algorithm to minimize the chi-squared unless otherwise specified. Summarizing different models and genes, the following kinetic rates were estimated: transcript elongation rate, intron removal rate, nuclear export rate, nuclear degradation rate, cytosolic degradation rate, cytosolic transport rate and membrane degradation rate.
Assembly: mm10
Supplementary files format and content: {sample}.txt files provide raw read counts per gene, reads counted in exons with HTSeq-count
Supplementary files format and content: {sample}_t2c_counts.txt files give gene and exon-wise T2C metrics: conversion probability (p_conv), number of Ts in gene/exon/intron (T_exon, T_intron), number of T2C transitions in gene/exon/intron (T2C_exon, T2C_intron) and share of T2C vs total T counts divided by conversion probability per gene/exon/intron (T2C_over_T_exon, T2C_over_T_intron).
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| Submission date |
Dec 28, 2023 |
| 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) |
| GSE252199 |
Quantification of subcellular mRNA kinetics in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN39181841 |
| SRA |
SRX23052567 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7996485_c0min3.txt.gz |
200.9 Kb |
(ftp)(http) |
TXT |
| GSM7996485_c0min3_t2c_counts.txt.gz |
2.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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