GSM5914795: RNAcapture for ts-ARFGEF1; Homo sapiens; OTHER
To capture the transcript of ts-ARFGEF1, we performed a TERRA-capture RNA experiment as previously described. (PMID: 28692038) 20 g of Trizol-purified total RNA was treated with 4 U of TURBO DNase (Thermo Fisher Scientific, Cat. AM2238) at 37 oC 10 min in 100l with 1 l RNaseOUT (Thermo Fisher Scientific, Cat. 10777019). The RNA was purified by Trizol. 20 g RNA was mixed with 10 pmol of biotin-labeled oligo probes against NCL region of ts-ARFGEF1 (TCCAAGCCAACTGTTCCGTT/3'BioTEG) or scramble negative control probe (GAATTCGTCCTCCGATTCAC/3'BioTEG), denatured at 70 oC in 100 l6×SSC hybridization buffer for 10 min and then lowered the incubation temperature to 44 oC (0.5 oC/s). RNA was then captured by 100 l Dynabeads MyOne Streptavidin C1 (ThermoFisher, Cat. 65001) and washed with 2×SSC/0.1% NP40 at 37 oC 5 min for 4 times, washed twice with 1×SSC/0.1% NP40 at 37 oC, and rinsed once with 1×SSC at room temperature. After serial wash steps, the captured RNA was eluted by 30 l DEPC treated water at 70 oC for 5 min. 3 l elution was used for the analysis of capture efficiency. After capturing the transcript of ts-ARFGEF1 by biotin-labeled probe against NCL junction, captured RNA was used for generating cDNA for nanopore sequencing according to the ONT cDNA-PCR Sequencing protocol by the manufacturers (Oxford Nanopore Technologies, SQK-PCS109). Complementary strand synthesis from RNA and strand switching was conducted by using kit-supplied oligonucleotides. Double strand cDNA is then generated by PCR amplification using primers that contain 5' tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters. Finally, three libraries with ~11 ng PCR products were sequenced by an R9.4 Flow cell with three times priming.