All RNA extracted from wild-type cells inclusing HepG2, HeLa and HEK293T cell lines. Basically, as the first step of harvesting cells, the cells were washed once with DPBS buffer for each plate after aspirating the media. Then cellular total RNA was isolated with TRIzol reagent (Invitrogen) following manufacturer's protocol by isopropanol precipitation. The small RNA fraction (size less than 200 nt) was further extracted from the prepared cellular total RNA by mirVana™ miRNA Isolation Kit (AM1560, Invitrogen) protocols. Around 300 ng human cellular small RNA (size less than 200 nt) was fragmented into 40-50 nt using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer's protocol and Oligo Clean & Concentrator (Zymo Research). Save ~50 ng as “input” and perform enhanced demethylation treatment towards the rest fragmented RNA with 1.0 uL AlkB mutant (10 mg/mL protein concentration), followed by purification using Oligo Clean & Concentrator (Zymo Research). Then the “input” RNA and demethylase treated RNA were sent for end repair mediated by T4 Polynucleotide Kinase (EK0032, Thermo Fisher Scientific): RNA was mixed with 5 uL 10X T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB) and 5 uL T4 PNK, diluted to a final volume of 50 uL and incubated at 37 °C for 1 hour followed by Oligo Clean & Concentrator (Zymo Research). Starting with 3'-adaptor ligation, repaired RNA fragments were incubated with 1.0 uL 50 uM 3'-linker (5'App-AGATCGGAAGAGCGTCGTG-3SpC3) at 70 °C for 2 min and transferred to ice immediately. 2,5 uL 10X T4 RNA Ligase Reaction Buffer (NEB), 7.5 uL 50% PEG8000, 1 uL SUPERase•In™ RNase Inhibitor and 2 uL T4 RNA ligase 2 truncated KQ (NEB) were added accordingly to the RNA-adapter mixture, then the reaction was diluted to a final volume of 25 uL and incubated at 25 °C for 2 hours followed by 16 °C for 12 hours. The completed ligation reaction was further diluted into 47 uL by Nuclease-free water, and the excessive adapters were removed with 2 uL 5'-Deadenylase (NEB) at 30 °C for 1 hour followed by adding 1.0 uL RecJf (NEB) for ssDNA digestion at 37 °C for 1 hour. The 3'-end ligated RNA was extracted by RNA Clean & Concentrator-5 (Zymo Research). The purified RNA was incubated with 1.0 uL of 2.0 uM RT primer (5'-ACACGACGCTCTTCCGATCT-3') at 65 °C for 2 min and moved onto ice immediately. Then 2 uL 10X AMV Reverse Transcriptase Reaction Buffer (NEB), 2 uL 10 mM dNTP Solution Mix (NEB), 1 uL RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific) and 2 uL HIV Recombinant Reverse Transcriptase (Worthington) were added into the RNA-primer mixture, followed by a one-hour incubation (diluted into 20 uL volume) at 37 °C for 90 min. The RT enzymes were deactivated at 70 °C for 5 min then kept on ice. The harvested cDNAs were purified and collected by Oligo Clean & Concentrator (Zymo Research). Then the mixture of cDNA and 1.0 uL of 50 uM cDNA 3'-linker (5'Phos-NNNNNNAGATCGGAAG AGCACACGTCTG-3SpC3) were denatured at 75 °C for 2 min and then put onto ice. 2.5 uL 10X T4 RNA Ligase Reaction Buffer (NEB), 12.5 uL 50% PEG8000, 2.5 uL 10 mM ATP and 1.0 uL T4 RNA ligase 1 (high concentration, NEB) were added accordingly to cDNA-adapter mixture, then the reaction was incubated at 25 °C for 12 hours. Finally, the libraries were amplified using the universal and indexed primers from NEBNext Multiplex Oligos for Illumina (NEB). All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.
GEO Accession
GSM4456045