GSM5917184: Positive-AGS_FN1_3UTR; Homo sapiens; miRNA-Seq
RNA pulldown assays were performed with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher) according to the instructions. In vitro transcribed (IVT) RNA probes for pull-down assays were prepared with AmpliScribe™ T7 High Yield Transcription Kit (Epicentre). In brief, 10^7 cells were collected and washed in ice-cold phosphate-buffered saline. The cell pellets lysed in 1ml IP lysis buffer, and centrifuged at 12000 g for 15 minutes at 4°C to collect the supernatant. Second, 50 μl washed streptavidin magnetic beads incubated with 5 μg biotinylated IVT lncRNA or its antisense RNA for 30 minutes at room temperature with agitation. Then, probes coated beads incubated with 500 μl cell lysis supernatant for 1 hour. The beads were washed briefly with wash buffer for five times and elutioned. The bound protein or RNA in the pull-down materials were purified for further analysis. High-throughput sequencing service was provided by CloudSeq Biotech (Shanghai, China). Total RNA of each sample was used to prepare the miRNA sequencing library, which included the following steps: 1) 3'-adaptor ligation; 2) 5'-adaptor ligation; 3) cDNA synthesis; 4) PCR amplification; 5) Size selection of ~150 bp PCR amplicons (corresponding to ~22nt miRNAs). The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 50 cycles on Illumina Novaseq 6000 sequencer following the manufacturer's instructions.