2025-09-14
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114328120
External Id
Dementia Research Institute at KCL, London, UK The Francis Crick Institute, London, UK National Institute of Chemistry, Ljubljana, Slovenia
INSDC center name
2023-09-05T10:08:09Z
INSDC last update
public
INSDC status
ERS16317230
SRA accession
E-MTAB-13331:Srrm2_high_2
Submitter Id
ArrayExpress
broker name
mouse embryonic stem cell
cell_type
not collected
collection date
house mouse
common name
Protocols: mESCs were grown in 10cm dishes to ~70% confluency, crosslinked with 200mJ/cm2 254nm UV and cell pellets were snap frozen. mESCs were grown under 2i conditions feeder free, with passaging every 2-3 days and daily media exchanges. 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. Antibodies against GFP (ab290) coupled to magnetic Protein G beads were used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labelled IRL3 adaptor. The complexes were then size-separated by SDS-PAGE, blotted onto nitrocellulose and visualised by Odyssey scanning. 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.For the ‘RNase experiment’ we use 4 units Turbo DNase and 0.4U RNase I per 1 ml of lysate at 1mg/ml protein concentration. 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.
description
embryo stage
developmental stage
normal
disease
Srrm2-GFP knock-in
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
blastocyst
organism part
Mus musculus
scientific_name
Srrm2_high_2
title