GSM9742973: RBE cell, NC_1; Homo sapiens; RNA-Seq
Total RNA was extracted using chloroform. mRNA with poly(A) tails was enriched using Oligo(dT)-conjugated magnetic beads. The obtained RNA was fragmented with fragmentation buffer, and reverse transcription was performed using random N6 primers, followed by second-strand synthesis to generate double-stranded cDNA. The double-stranded cDNA was end-repaired, 5'-phosphorylated, and a single adenine (A) overhang was added at the 3' ends. Bubble-shaped adapters with a protruding thymine (T) at the 3' ends were then ligated. The ligation products were amplified by PCR using specific primers. The PCR products were thermally denatured into single strands, and a bridging primer was used to circularize the single-stranded DNA to form a single-stranded circular DNA library. The constructed library was quality-checked and subjected to sequencing upon passing the quality assessment.