GSM7688020: S21; Homo sapiens; ncRNA-Seq
For isolation of total RNA, the miRNeasy Mini kit (Quagen, cat. No. 217004) was used according to the manufacturer's protocol Ribosomal RNA depletion was performed using the Ribozero HMR kit (Illumina). The rRNA-depleted RNA was purified by 2x RNAClean XP beads (Beckman Coulter) and eluted in 45µL of nuclease-free water. 5µL of purified rRNA-depleted RNA was mixed with 4µL of NEBNext First Strand Synthesis Reaction Buffer and 1µL of random primers. The reaction was incubated at 94°C for 12min for fragmentation. Subsequently, to perform first strand cDNA synthesis, 8µL of NEBNext Strand Specificity Reagent and 2µL of NEBNext First Strand Synthesis Enzyme Mix was added to the reaction and the reaction was incubated at 25°C for 10 mins, 42°C for 30 mins, 70°C for 15 mins. Second strand reaction was then performed by adding 8µL of NEBNext Second Strand Synthesis Reaction Buffer with dUTP Mix (10X), 4µL of NEBNext Second Strand Synthesis Enzyme Mix, and 48µL of nuclease-free water. The reaction was incubated at 16°C for 1h. The cDNA was purified by 1.8x SPRIselect Beads (Beckman Coulter) and eluted in 50µL of nuclease-free water. Subsequently, endprep reaction was performed by adding 7µL of NEBNext Ultra II End Prep Reaction Buffer and 3µL of NEBNext Ultra II End Prep Enzyme Mix into 50µL purified cDNA. Endprep reaction was incubated at 20°C for 30 mins and 65°C for 20 mins. The adaptor ligation reaction was then performed by adding 1µL of NEBNext Ligation Enhancer, 30µL of NEBNext Ultra II Ligation Master Mix, and 2.5µL of NEBNext Adaptor, diluted to 0.5µM in Adaptor Dilution Buffer. The mix was incubated at 20°C for 15min. 3µL of USER Enzymer (New England Biolabs) was then added to the ligation product and the reaction was incubated at 37°C for 15min. The ligated product was purified by SPRIselect Beads (Beckman Coulter) and eluted in 15µL of nuclease-free water. PCR was carried out by adding 25µL of NEBNext Ultra II Q5 Master Mix, 5µL of i5 Primer, and 5µL of i7 Primer into 15µL of purified ligated product. PCR was performed at 98°C for 30s, 15 cycles of 98°C for 10s and 65°C for 75s, and a final extension at 65°C for 5min.  The final library was then purified by SPRIselect Beads.