Lysis of extracellular vesicle and dissociation of ribonucleoprotein
Library construction starts with 3-7 µL serum sample. Ultrapure water is firstly added to make a total volume of 7 µL. Lysis of extracellular vesicle and dissociation of ribonucleoprotein are then performed by thoroughly mixing the sample with 1.7 µL 11.5 mM DTT solution, 0.5 µL 40 U/µL RNase inhibitor and 2.8 µL lysis buffer consisting of 10 mM Tris-HCl, 0.2 % w/v SDS solution and 4 % w/v NP-40.
First-strand cDNA synthesis
Random and oligo-dT primers are added to the sample, the mixture is then incubated at 70°C for 2 min. Afterward, HL-dsDNase is added to the sample, the mixture is then incubated at 37°C for 10 min for genomic DNA digestion and at 65°C for 5 min for enzyme deactivation. Finally, reverse transcriptase is added, and the first-strand cDNA is synthesized with a series of incubations, 25°C for 5 min, 40°C for 30 min and 70°C for 10 min.
Second strand cDNA synthesis
Firstly, the cDNA is processed with steps such as fragmentation. DNA polymerase and template-switching oligo are then added, and the second strand cDNA is synthesized with a series of incubations, 25°C for 15 min, 37°C for 15 min and 70°C for 10 min.
Library processing and amplification
End repair and adaptor ligation are performed, followed by purification and size-selection with 1× volume of AMPure XP beads. Library is then amplified with PCR, followed by purification and size-selection with 0.8× volume of AMPure XP beads. Subsequently, depletion of rRNA sequences is carried out. Firstly, probes targeting rRNA sequences, DNA polymerase, etc. are added, the mixture is incubated at 37°C for 10 min, 95°C for 2 min, 50°C for 1 min and 65°C for 10 min to form a cleavage site in the adaptor region of cDNA molecules containing rRNA sequences. Then, restriction enzyme is added, the mixture is incubated at 55°C for 30 min, 95°C for 5 min to break the cleavage site to make the cDNA molecules not amplifiable. Afterwards, another PCR amplification is conducted, relative abundance of cDNA molecules containing rRNA sequences is minimized while other cDNA molecules are exponentially amplified. At last, library is purified and size-selected again with 1× volume of AMPure XP beads.
The product library is quantified with Qubit and measured by Bioanalyzer or Fragment Analyzer for size distribution.