title: GSM3764530: A549 lipofectamine only; Homo sapiens; OTHER
library_construction_protocol: Total RNA was purified using the RNeasy Kit (Qiagen) RtcB RNA-seq was conducted as described previously (Donovan et al., 2017), but without short RNAs purification step. Briefly, 1 µg RNeasy-purified RNA was ligated to 10 µM adaptor (Table 1, oligo 1). Ligation reactions were performed using 10 µM RtcB, 20 mM HEPES pH 7.5, 110 mM NaCl, 2 mM MnCl2, 100 µM GTP, 40U RiboLock RNase inhibitor (Fermentas), 4 mM DTT, and 0.05% Triton X-100 for 1 hour at 37 °C. Reactions were quenched using 3 mM EDTA. Owing to its short length, free adapter was removed by purifying the ligated RNA with the RNeasy kit. On-column DNase treatment was omitted so that ligated adapter remained intact. Oligonucleotides were reverse-transcribed using MultiScribe reverse transcriptase (RT) and 2 pmol of RT primer complimentary to the ligation adaptor (Table 1, oligo 2). RNA, RT primer, and dNTPs were incubated for 3 min at 65°C and snap-cooled on ice. MgCl2 (3 mM f/c) was added to ensure efficient Mg2+-dependent RT reaction. A 2x mastermix containing RT buffer, RT (Applied Biosystems), and 40U Ribolock was added to snap-cooled samples to a final volume of 20 µL. Reactions were incubated at 25°C for 10 min, 37°C for 1.5 h, and 95°C for 5 min. cDNA reaction was brought up to 200 µl with water and extracted using 1 volume of 25:24:1 Phenol:Chloroform:Isoamyl Alcohol saturated with 10 mM TRIS (pH 8.0) and 1 mM EDTA (Sigma Aldrich). The aqueous phase was precipitated using 20 µg glycogen as a carrier, 2/3 5M ammonium acetate (vol/vol) and 3 volumes of 100% ethanol. Mixture was incubated at –80 °C for 30 minutes, followed by a 17,000 x g spin at 4 °C, for 30 minutes. Pellets of cDNA were washed with 75% ethanol (vol/vol) and resuspended in 25 µl DI water. Reactions contained 30% of the cDNA from the previous step, 1 µM adaptor (Table 1, oligo 3), 1 U/µl CircLigase (Epicentre), and buffer contents as per manufacturers guidelines. CircLigase reactions were incubated for 1 h at 65°C and quenched by adding EDTA to a final concentration of 8 mM. 1/3 of the quenched CircLigase reaction was PCR amplified using Phusion DNA polymerase (NEB) and primers 4-5 in Table 1. Libraries were analyzed by a BioAnalyzer high sensitivity DNA 1000 chip (Agilent). Equimolar amounts were pooled and gel purified from native page. RtcB RNA-seq was performed on an Illumina HiSeq 2500 and processed as described previously (Donovan et al., 2017).