title: GSM3384011: IDH1 FBioCLIP-seq-2; Mus musculus; OTHER
library_construction_protocol: UV-crosslinked cells were lysed in ice-cold Lysis Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 5% glycerol, supplemented with 1 mM DTT, 1 mM PMSF, 1:500 PI cocktail and 400 U/mL RNase Inhibitor), tandem immunoprecipitation, fragmented RNA transcripts were purified using TRIzol according to manufacturer's protocol. The pre-adenylated 3' linker was ligated to the end of fragmented RNAs with T4 RNA ligase (NEB) at 16 °C overnight. Then, after four times washes with PNK Buffer, the bead-bound RNA fragments were phosphorylated by T4 PNK (NEB) for 10 min at 37 °C and the reaction was stopped by washing three times with PNK-EGTA Buffer. The protein-bound RNA fragments were eluted by Proteinase K treatment. The purified RNAs were ligated with 5' RNA adaptor. The RNA fragments were then reverse transcribed by Superscript III (Invitrogen) with RT primer and amplified for 20 cycles with 2 × NEB HF PCR mix (NEB). Library index sequence was introduced by PCR with index primers for 8 more cycles.
identifiers.namespace: GEO
identifiers.id: GSM3384011