title: GSM2935049: TRMT2A_HEK293_FICC-seq_rep2; Homo sapiens; RIP-Seq
library_construction_protocol: For FICC-Seq experiments, cells were treated with 100µM 5-FU (Sigma) for 24 hours and then harvested. Cell pellets were disrupted in lysis buffer consisting of 50 mM Tris-HCL pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and treated with Turbo DNase (AM2239).  Lysates were also treated with a low concentration of RNaseI (Thermofisher AM2295 at 1:200 dilution) in order to partially fragment RNAs. Lysates were cleared by centrifugation at 13,000 r.p.m. for 15 min at 4°C and then incubated with Protein G Dynabeads (Life Technologies) in the presence of anti-TRMT2A antibody (Origene, OTI1C3). Following stringent washing, 3′-end dephosphorylation was performed with T4 PNK (New England Biolabs) before addition of a preadenylated linker using RNA ligase (New England Biolabs). 5′-end labelling was then performed using T4 PNK and 32P-ATP before protein–RNA complexes were eluted and run on Bis-Tris polyacrylamide gels. Next, nitrocellulose transfer was performed and the radioactive signal was used to dissect nitrocellulose pieces that contained TRMT2A-RNA complexes. RNA was recovered by incubating the nitrocellulose pieces in a buffer containing Proteinase K and 3.5 M urea. Reverse transcription was performed using oligonucleotides containing randomized barcodes (UMIs) and two inversely oriented adaptor regions separated by a BamHI restriction site. cDNAs were size-purified on TBE-Urea gels before being circularized by CircLigase II (Epicentre). Circularized cDNAs were then annealed to an oligonucleotide complementary to the BamHI site and then BamHI digested. Linearized cDNAs were then PCR-amplified using primers complementary to the adaptor regions using 25 cycles of PCR. Libraries containing in-line barcodes were then sequenced on the Illumina HiSeq4000 platform. Cells were harvested by washing in Phosphate Buffered Saline (PBS) and then incubating with Trypsin-EDTA followed by further washing of detached cells in PBS.
attributes.tag: GEO Accession
attributes.value: GSM2935049