GSM9448789: MIO-M1 cells, siControl for siMapk14_2, Day 3; Homo sapiens; RNA-Seq
Total RNA was extracted from cells or retinal tissues with  TRIzol reagent. Following assessment of quality, samples with an RNA integrity number (RIN) greater than 7.5​ were selected for library construction. Capture mRNA with polyA tails using magnetic beads conjugated with Oligo(dT). RNA Fragmentation and Reverse Transcription:​ Fragment the obtained mRNA using a fragmentation buffer. Perform reverse transcription with random N6 primers to synthesize the first cDNA strand, followed by second-strand cDNA synthesis to generate double-stranded DNA. Double-Stranded DNA Processing and Ligation:​ Blunt-end and phosphorylate (5'-end) the double-stranded DNA, and create a 3'-end overhang with a single adenine ("A") base. Then ligate a bubble-shaped adapter that has a 3'-end overhang with a single thymine ("T") base. PCR Amplification and Circularization:​ Amplify the ligation product by PCR using specific primers. The PCR product is then heat-denatured into single strands. A bridge primer is used to circularize the single-stranded DNA, resulting in a single-stranded circular DNA library. Library Quality Assessment and Sequencing:​ The constructed library undergoes quality control. Once it passes quality inspection, it is ready for sequencing.