GSM6654464: RNA D3-delbelt-G1-G4-dimer products set1; synthetic construct; OTHER
Illumina adapter sequences were incorporated to the libraries using PCR. The purified F3 fragments were ligated to 10 pmol 5ꞌ-adapter (RA5-6N, GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN NNN) in a reaction mixture containing 1 μL T4 RNA ligase I (NEB, M0204L), 2 μL 10x T4 RNA ligase buffer, 6 μL of 50% PEG8000, and 0.5 μL of SUPERase•In RNase inhibitor (20 U/μL). The reaction was carried at 25°C for 16 h. The RA5-ligated RNAs were reverse-transcribed with R-RA3 primer (TTG GCA CCC GAG AAT TCC A) by Superscript IV reverse transcriptase (Invitrogen, 18090050). The resulting cDNA was PCR-amplified using a pair of sequencing primers RP1 and one of RPIx.   The original substrates were  reverse-transcribed with R-RA3 primer by Superscript IV reverse transcriptase (Invitrogen, 18090050). The cDNA was first PCR-amplified using a pair of primers F-RA5-18-comp (TTC AGA GTT CTA CAG TCC GAC GAT CCT GCC ATT TTA CAA TCC A) and one of RPIx. The resulting DNA was amplified using a pair of sequencing primers RP1 and one of RPIx.