GSM8888139: Non cancer control 5 (Pancretic cancer cohort); Homo sapiens; RNA-Seq
cfRNAs were isolated from 600 μl plasma using QIAamp ccfDNA/RNA Kit (Qiagen, Cat. 55184) according to the manufacturer's protocol. In brief, 180 μl RPL buffer was added into 600 μl plasma, followed by vortexing for 5 s. After leaving at room temperature for 3 mins, 100 μl RPP buffer is added. Then close EP tube cap and immediately mix vigorously by vortexing 10 s for 2 times, which is important to disperse the solid. Incubate the EP tube on ice for 3 min. After the standard steps of washing and concentration following the manufacturer's protocol, the purified cfRNA was finally eluted in 14 µl of RNase-free water. Plasma cell-free RNA (cfRNA) or small RNA from HepG2 (<200 nt) were fragmented to lengths of ~35-55 nucleotides using the 10X RNA Fragmentation Reagent (AM8740, Invitrogen), which was used as 15X with heating at 70 °C for 14 mins. Purify the fragmented RNA using the Oligo Clean & Concentrator kit (Zymo Research) with a final elution volume of 10 μl. The subsequent 3′-end repair involved the preparation of a mixture of fragmented RNA, 2 μl of 10× T4 Polynucleotide Kinase Reaction Buffer (B0201S, NEB), 2 μl of T4 PNK, 1 μl SUPERase•In RNase Inhibitor and a proper volume of RNase-free water. Mix the 20 μl reaction mixture very well and incubate at 37°C for 1 hour. 3'-end-repaired RNA fragments were purified by Oligo Clean & Concentrator kit and eluted in 10 μl of RNase-free water. For 3′-adapter ligation, RNA fragments were denatured with 1ul of 10 μM RNA 3′-linker (5′rApp-NNNNN AGATCGGAAGAGCGTCGTG-3SpC3) by heating at 70°C for 2 minutes and then immediately moved onto ice. Then a mixture of 2.5 μl of 10× T4 RNA Ligase Reaction Buffer (NEB), 7.5 μl of 50% PEG8000, 1 μl SUPERase•In, and 2 μl T4 RNA ligase 2 truncated KQ (NEB) was prepared, followed by mixing well and incubating at 25°C for 2 hrs and then at 16°C for 10 hrs. The excessive adapters were removed by adding 2 μl of 5′-deadenylase (NEB) with an incubation at 30 °C for 45 mins, followed by adding 1.0 μl of RecJf (NEB) with an incubation at 37 °C for 45 mins. The 3'-ligated RNA was then purified by RNA Clean & Concentrator (Zymo Research) and eluted in 10 μl of RNase-free water. Reverse transcription was initiated by mixing the 3'-ligated RNA with 1 ul of 2.0 μM RT primer (5′-ACACGACGCTCTTCCGATCT-3′) and heating at 65°C for 2 minutes. Then move the samples onto ice immediately. A mixture of 2 μl of 10× AMV Reverse Transcriptase Reaction Buffer (NEB), 2 μl of 10 mM dNTPs, 1 μl RNaseOUT (Thermo Fisher Scientific), 2 μl HIV Recombinant Reverse Transcriptase (Worthington) and a proper volume of RNase-free water was prepared, to give a final volume of 20 μl for a one-hour incubation at 37°C. Upon the completion of RT, 1.0 μl volume of RNase H (NEB, M0297L) was added to the reaction mixture, followed by incubation at 37 °C for 20 min and 70 °C for 5 min. The produced cDNAs after RT were purified by Oligo Clean & Concentrator (Zymo Research) and eluted in 10 μl of RNase-free water. A mixture of eluted cDNA and 2.0 μl of 10 μM cDNA 3′-linker (5′Phos-NNNNNAGATCGGAAGAGCACACGTCTG-3SpC3) was denatured at 75 °C for 2 min and then moved onto ice immediately. 3-μl of 10× T4 RNA Ligase Reaction Buffer (NEB), 15 μl of 50% PEG8000, 3 μl of 10 mM ATP, and 1.0 μl of T4 RNA ligase 1 (high concentration, NEB) were added to the cDNA–adapter mixture and mixed very well. Incubate the ligation reaction mixture at 25 °C for 12 h. The libraries were amplified using universal and indexed primers from NEBNext Multiplex Oligos for Illumina (NEB). All libraries were sequenced on an Illumina NovaSeq 6000 or NovaSeqX with SE100 bp mode. The LIME-seq method enables high mutation ratio detection across multiple RNA modifications, including m1A, m3C, m1G, m22G, m3U, etc., with an enhanced read-through at these methylation sites. Such performance has been facilitated by the robust HIV RT enzyme, and it effectively uncovered methylation fraction information at methylated sites