GSM6381769: HeLa_SmallRNA_HIV-RT_Treated_rep2; Homo sapiens; RNA-Seq
Cellular total RNA was isolated with TRIzol reagent (Invitrogen) following manufacturer's protocol by isopropanol precipitation. Then, cellular small RNAs (<200 nt) were extracted by mirVana™ miRNA Isolation Kit following manufacturer's protocol. The starting amount of cellular small RNA (< 200 nt) could be 200 ng before fragmentation. Then Zinc RNA Fragmentation Rea-gents (Invitrogen, AM8740) was used as 15X (add 1.0 µL buffer into 14 µL of RNA), which is originally 10X. The heating condition is always 70 °C for 14 min, followed by Oligo Clean and Concentrator kit (OCC). Elute RNA to 22 µL (twice, 11 µL each time). Prepare a stock containing 22 µL RNA + 3 µL 10X PNK buffer + 3 µLT4 PNK + 1.5 µL SUPERase•In™ with a final volume of 30 µL. Mix the stock well and incubate at 37 °C for 1 hour. Recover RNA with Oligo Clean and Concen-trator kit (OCC) and elute to 10 µL with RNase-free water. 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 12 µL with RNase-free water. Save 2 µL as 'input'; use the rest for the following steps. Put the eluted RNA in 10 µL RNase-free water. Prepare a fresh 1.0M KBH4 buffer in RNase-free water. Add 40 µL KBH4 buffer into RNA and mix well. Incubate at room temperature for 2 hours (avoid the light). Recover RNA with RNA Clean and Concentrator kit (RCC) and elute to 45 µL with RNase-free water. Add 5.0 µL 1.0M NaOAc/AcOH buffer pH 2.9 into the purified RNA and mix well. Incubate with heating for 4 hours (avoid the light). Recover RNA with Oligo Clean and Concentrator kit (OCC) and elute to 10 µL with RNase-free water. Mix 1.0 µL 2 uM RT-primer with 10 µL depurinated 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 2.5 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.