GSM8678760: PAC-1; Mus musculus; ncRNA-Seq
Total RNA was extracted from the samples, and ribosomal RNA was removed to maximize the retention of both coding and non-coding RNAs. The resulting RNA was randomly fragmented into short fragments. These RNA fragments were used as templates to synthesize the first strand of cDNA using random hexamer primers. Subsequently, buffer, dNTPs (with dUTP replacing dTTP), RNase H, and DNA polymerase I were added to synthesize the second strand of cDNA. The double-stranded cDNA was then purified using a QiaQuick PCR kit and eluted with EB buffer.After end repair and addition of an adenine (A) base to the 3' ends, sequencing adapters were ligated. The second strand of cDNA was selectively degraded using Uracil-N-Glycosylase (UNG) to retain only the strand with dUTP incorporation. Size selection of the fragments was performed using agarose gel electrophoresis, followed by PCR amplification.