GSM6381767: CalibrationCurves_HIV-RT_100%_AP-site; synthetic construct; RNA-Seq
Synthetic RNA oligo probes were used for library construction. For 200 ng of each mixed RNA oligo set (100%, 80%, 60%, 40% ,20% and 0% AP-site), proceed with RNA 3'ligation directly. Mix 1.0 µL 20 µM 3' linker (5'rApp + random-barcode + adaptor sequence + C3-spacer) with repaired 10 µL RNA fragments and incubate the mixture at 70 °C for 2 min for denaturation. Immediately move onto ice. Then add 2.5 µL 10X T4 RNA Ligase Rxn Buffer, 7.5 µL 50% PEG8000, 1 µL SUPERnaseIn, and 1 µL H2O. Mix everything well before adding T4 RNA ligase 2 truncated KQ. Then add 2.0 µL T4 RNA lig-ase 2 truncated KQ and mix well again. Incubate the reaction at 25 °C for 2 hours, followed by 16 °C for 12 hours. Then add 23 µL RNase-free water to dilute the reaction mixture. Add 2.0 µL 5-deadenylase, mix well/incubate at 30 °C for 30 min; then add 1.0 µL RecJf, mix well/incubate at 37 °C for another 30 min. Recover RNA with RNA Clean and Concentrator kit (RCC) and elute to 10 µL with RNase-free water. Mix 1.0 µL 2 uM RT-primer with 2 µL ligated RNA fragments and incubate the mixture at 65 °C for 2 min for denaturation. Immediately move onto ice. Then add 2 µL 10X AMV RT Rxn Buffer, 2 µL 10mM dNTP, 0.5 µL RNaseOut, and 10.5 µL H2O. Mix everything well before adding HIV RT. Then add 2.0 µL HIV RT and mix well again. Incubate the reaction at 37 °C for 1.5 hours. After RT, add 1.0 µL RNaseH (NEB) into the reaction mixture. Mix well/incubate at 37 °C for 20 min. Then 70 °C for 5 min to denature. Recover RNA with Oligo Clean and Concentrator kit (OCC) and elute to 10 µL with RNase-free water. Mix 1.0 µL 50 uM cDNA 3'-adapter with 10 µL cRNA and incubate the mixture at 75 °C for 2 min for denaturation. Immediately move onto ice. Then add 3 µL 10X T4 RNA Ligase Rxn Buffer, 15 µL 50% PEG8000, and 3 µL 10mM ATP. Mix everything well before adding T4 RNA Ligase 1. Then add 1.0 µL T4 RNA Ligase 1 and mix well again. Add ligase enzyme to one sample followed by mixing this sample immediately. Mix each sample one by one. Do not add ligase enzyme to all samples and then start mixing everything.  Incubate the reaction at 25 °C for 12 hours. After the 12-hour cDNA ligation, heat at 65 °C for 5 min for denature. Purify the cDNA by DNA Clean & Concentrator-5. Elute cDNA with 20 µL DNase-free water the reaction mixture. Mix again and then use 4 µL per sample for each PCR amplification. RNA-Seq. All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.