GSM5209148: lin28a-gfp-esc-naive7; Mus musculus; RIP-Seq
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