GSM4190647: Untransfected control_bind; synthetic RNA; OTHER
The interacted RNA was isolated by phenol-chloroform extraction and ethanol precipitation. The RNA pellet was suspended in 24 µl of nuclease-free water and subjected to RT with SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, #18090010). Prior to reverse transcription, 20 µL of purified RNA library was mixed with 1 µL of 10 µM reverse primer (5'-GCGTCCATGGTCGACCTGAGGTCC-3'), 1 µL of 10 mM dNTPs, and 4 µL of nuclease water, and then denatured at 65°C for 5 minutes. The mixture was combined with a premix of 8 µL of 5X SSIV Buffer, 2 µL of 100 mM DTT, 2 µL of RNase OUT (Thermo Fisher Scientific, #10777019), and 2 µL of SuperScript IV Reverse Transcriptase. To prepare an input random RNA library, 0.5 pmol of input random RNA library was reverse transcribed. After digestion of RNA with 2 µl of RNaseH for 20 min at 37°C, the cDNA preparation was amplified by PCR in a reaction mixture containing 1× Platinum SuperFi PCR Master Mix, 5 µM forward primer (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGACGTGACACGACGTGC-3'), 5 µM reverse primer (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGCGTCCATGGTCGACCTGAGGTCCGACG-3'), 1× SuperFi GC Enhancer, and 2 µl of cDNA and adjusted to 25 µl with nuclease-free water. The incubation protocol included an initial denaturation at 98°C for 30 s, 20 cycles of denaturation at 98°C for 10 s and extension at 72°C for 10 s, and a final extension at 72°C for 5 min. Single-stranded DNA was digested with Illustra ExoProStar exonuclease I (GE Healthcare Life Sciences, #US78211), and the PCR products were purified with the use of a MinElute PCR Purification Kit (Qiagen, #28006) before the addition of Illumina sequencing barcodes by 10 cycles of PCR with index primers from a Nextera XT Index Kit (Illumina, #FC-131-1001). The reaction mixture contained 1× Platinum SuperFi PCR Master Mix, 5 µl of Index 1 (i7) primer, 5 µl of Index 2 (i5) primer, 1× SuperFi GC Enhancer, and at most 5 µl of the PCR products, and the volume was adjusted to 50 µl with nuclease-free water. The incubation protocol included an initial denaturation at 98°C for 30 s; 10 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and extension at 72°C for 20 s; and a final extension at 72°C for 5 min. The resulting sequencing library was quantified with the use of a KAPA Library Quantification Kit (Illumina) Universal qPCR Mix (Kapa Biosystems, #07960140001). The library was sequenced for 150 cycles on the Illumina MiSeq platform and with a MiSeq Reagent Kit v3 (Illumina, #MS-102-3001).
GEO Accession
GSM4190647