GSM8172110: HeLa cells, sh-Luc-1; Homo sapiens; RNA-Seq
Total RNA was isolated from the pellets (1×107 cells) using Ambion TRIzol® Reagent according to the manufacturer's instructions, followed by DNase I treatment at 37°C for 60 min. Ligation reactions contained 2.0 µg total RNA, 1×T4 RNA ligase buffer, 5% PEG8000, 1 U T4 RNA ligase I, and 5 μM 3′ linker (CKOligo-666). The ligation reaction mixture was incubated at 16°C for 18 hours and inactivated at 65°C for 15 minutes. 20 μl of reverse transcription reaction contained 1X ligation reactions, 1× SSIV buffer, 0.1 μM RT primer (CKOligo-667), 0.5 μM dNTPs, 5 mM DTT, RNase inhibitor, and SuperScript™ IV Reverse Transcriptase was incubated at 50°C for 10 minutes. A total of 10 U E. coli RNase H was directly added into the reverse transcription reaction, followed by incubation at 37°C for 20 minutes and then heat-inactivation at 70°C for 15 minutes. The first round PCR was performed with 0.5 μM CKOligo-667 and CKOligo-668 in 50 μl reactions containing 1× Phusion HF buffer, 0.2 mM dNTPs, and 0.02 U Phusion Hot Start II High Fidelity DNA polymerase. First-round PCR products were purified by PCR purification kit. For the second-round PCR reactions, 5 μl of first-round PCR product was used as a template. 0.5 μM CKOligo-669 as the forward primer for all samples and reverse primers CKOligo-676 (sh-Luc), CKOligo-685 (sh-PARN), CKOligo-686 (sh-LARP7), and CKOligo-687 (sh-MePCE) were used. Amplicons were purified by PCR purification kit (TOOLS).