iCLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs
iCLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs
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.