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Status |
Public on Mar 01, 2024 |
Title |
lin28a-gfp-episc-primed4 |
Sample type |
SRA |
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Source name |
ESC
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Organism |
Mus musculus |
Characteristics |
strain: mESC J1 developmental stage: epiblast stem cells group: Primed_group tag: CRISPR fusion of GFP to C-terminus of Lin28a gene
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Treatment protocol |
We used iCLIP to identify a repertoir of RBP binding sites in mESCs.
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Growth protocol |
mouse ESC lines were maintained in 1:1 Neurobasal (21103049) DMEM (11320033) medium containing N2 (17502048) and B27 (17504044) supplements, 1% Glutamax (35050), 1% nonessential amino acids (1140050), 0.1 mM 2-mercaptoethanol (31350-010) (all Thermo Fisher Scientific), 1 mM MEK inhibitor PD0325901 (1408, Axon Medchem), 3 mM GSK3 inhibitor CHIR99021 (4953/50 Tocris), and 1,000 U/ml LIF (ESGRO ESG1107, Merck), a condition named 2iLIF. Cells were passaged using Stempro-Accutase (A1110501, Thermo Fisher Scientific). Cells were grown on gelatin coated plates which were prepared by first incubating the wells 0.1% gelatin (EmbryoMax® ES-006-B, Merck) with 1% FBS (EmbryoMax® ES Cell Qualified FBS, ES-009-B, Merck) for 1 h at 37 °C followed by a rinse with PBS. Primed epiblast stem cells (EpiSCs) were cultured on gelatine and FCS-coated plates in N2B27 medium supplemented with recombinant 20 ng ml–1activin A (338-AC-050, R&D Systems) and 2 μM IWP2 (3533, Tocris) to suppress spontaneous differentiation. EpiSCs were passaged 1:4–1:10 every 3 days by triturating the colonies into small clumps using 0.5 mg ml–1 collagenase IV (Sigma). MEK/WNT-inhibited RSCs were cultivated with 1,000 U ml–1 LIF, 2 μM IWP2, 1 μM PD (manufacturers as mentioned above) and were passaged as ESCs.
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Extracted molecule |
total RNA |
Extraction protocol |
Cells were UV crosslinked on ice and then lysed in RIPA buffer. 0.4 Units of RNaseI and 4 Units Turbo DNase were added per 1 ml of cell lysate at 1mg/ml protein concentration for RNA fragmentation. Negative controls (no-UV) were prepared. Antibodies were coupled to magnetic Protein G beads used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labelled IRL3 adaptor (Zarnegar et al., 2016) with the following sequence: /5rApp/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA /iAzideN/AA AAA AAA AAA A/3Bio/ The complexes were then size-separated by SDS-PAGE, blotted onto nitrocellulose and visualised by Odyssey scanning. For the multiplexed sample 1 replicated was run in parallel with the IRL3 to allow quality control of the RNP complex on the membrane and to help with cutting of the bands. RNA was released from membrane by proteinase K digestion and recovered by precipitation. cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and AMPure XP beads purification (Beckman- Coulter, USA), then circularised using Circligase II (Epicentre) followed by AMPure XP beads purification. After PCR amplification, libraries were size-selected with Ampure beads (if necessary by gel-purification) and quality controlled for sequencing. Libraries were sequenced as single end 100bp reads on Illumina HiSeq 4000. iCLIP
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 4000 |
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Description |
Epiblast cells were grown for 12hours in grown , 20 ng ml–1 Activin A and 12 ng ml–1 bFGF. More details in the methods section listed in doi: https://doi.org/10.1101/2021.03.15.433780
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Data processing |
iCLIP reads were analysed on the iMaps server using the iCount software (https://github.com/tomazc/iCount). Briefly, experimental barcodes were removed (Martin, 2011) and sequencing reads aligned with STAR (Dobin et al., 2013) to mouse genome build (GRCm38.p5 GENCODE version 15 annotation) allowing two mismatches. Unique Molecular Identifiers (UMIs), were used to distinguish and remove the PCR duplicates. To determine protein-RNA contact sites, the sequencing read preceding nucleotide was assigned as the cross-link event. Significant contact sites were then identified, using the iCount function, based on false discovery rate (FDR) <0.05 comparing specific sites within a window of three nucleotides with randomised data (100 permutations) within co-transcribed regions. The significant cross-links signal was normalised by sequencing deep and million of tags (CPM). Then replicates were merged and summary of cDNA counts within genes and genic regions were generated with iCount segments function, normalising the counts by the length of the corresponding region. Genome_build: GRCm38.p5 GENCODE version 15 annotation Supplementary_files_format_and_content: Individual cross-link bed files, grouped cross-link bed files and peaks bedgraphs
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Submission date |
Mar 24, 2021 |
Last update date |
Mar 29, 2024 |
Contact name |
Jernej Ule |
E-mail(s) |
j.ule@ucl.ac.uk
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Organization name |
UCL Institute of Neurology
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Department |
Neuromuscular Diseases
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Lab |
RNA Networks Group
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Street address |
10-12 Russell sq
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City |
London |
ZIP/Postal code |
WC1B 5EE |
Country |
United Kingdom |
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Platform ID |
GPL21103 |
Series (2) |
GSE169547 |
Embryonic lumenogenesis is controlled by selective mRNA decay triggered by LIN28A relocation [iCLIP] |
GSE169555 |
Embryonic lumenogenesis is controlled by selective mRNA decay triggered by LIN28A relocation |
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Relations |
BioSample |
SAMN18474580 |
SRA |
SRX10437473 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5209138_lin28a-gfp-episc-primed4-20170607-mm_trimmed_single.bed.gz |
1.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
Processed data are available on Series record |
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