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Sample GSM5209138 Query DataSets for GSM5209138
Status Public on Mar 01, 2024
Title lin28a-gfp-episc-primed4
Sample type SRA
 
Source name ESC
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
Treatment protocol We used iCLIP to identify a repertoir of RBP binding sites in mESCs.
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.
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
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 4000
 
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
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
 
Submission date Mar 24, 2021
Last update date Mar 29, 2024
Contact name Jernej Ule
E-mail(s) j.ule@ucl.ac.uk
Organization name UCL Institute of Neurology
Department Neuromuscular Diseases
Lab RNA Networks Group
Street address 10-12 Russell sq
City London
ZIP/Postal code WC1B 5EE
Country United Kingdom
 
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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