GSM8889275: HCC95, siTP63b, rep2; Homo sapiens; RNA-Seq
Total RNA was isolated with TRIzol reagent (15596018, Invitrogen) from cultured cells For RNA-seq library preparation, poly (A)+ RNA was selected using the NEB-Next Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L) to perform the LM-seq procedure. including that isolated mRNA is fragmented in reverse transcription buffer with heat and then reverse-transcribed with SmartScribe reverse transcriptase (Clontech) using a random hexamer oligo (HZG883). After reverse transcription, RNA is removed by RNaseA and RNaseH treatment. A partial adaptor (HZG885) is then ligated to the single stranded cDNA using T4 RNA ligase 1 (NEB) and incubated overnight at 22℃. After purification, ligated cDNA is amplified by 18 cycles of PCR using oligos that contain full BGI adaptors. Last, the resultant libraries were purified using the DNA Clean Beads, followed by sequencing using the DNBSEQ-T7 PE150  platform  (Biopharmaceutical PublicService Platform, Nanjing).