GSM9421635: Rot3; Homo sapiens; RNA-Seq
Total RNA was extracted from experimental samples using TRIzol reagent (Thermo Fisher Scientific, San Diego, USA) according to the manufacturer's protocol. mRNA was enriched using poly(A) capture beads and purified. The enriched mRNA was fragmented by incubation at elevated temperature. First-strand cDNA synthesis was performed using fragmented mRNA as template in a reverse transcription system (Thermo Fisher Scientific, San Diego, USA). During second-strand synthesis, end repair and 3'-adenylation (A-tailing) were simultaneously completed. Illumina adapters were ligated to the cDNA fragments, followed by purification with Hieff NGS® DNA Selection Beads (Yeasen, Shanghai, China) for size selection. Libraries were amplified by PCR and quality-controlled.  ATAC-seq was performed according to established protocols. Briefly, 50,000 cells per sample were lysed to isolate nuclei. The nuclei were then extracted and incubated with Tn5 transposase (37°C, 30 min) for tagmentation. Illumina TruSeq adapters (N5 and N7) were ligated at a final concentration of 0.5 μM, followed by library amplification via PCR. Amplified libraries were purified using Novoprotein DNA Clean Beads (Novoprotein, Soochow, China). DNA concentration was quantified with a Qubit 2.0 Fluorometer (Thermo Fisher, MA, USA), and fragment size distribution was assessed using an Agilent 2100 Bioanalyzer. After quality control, 150-bp paired-end sequencing was conducted on an Illumina NovaSeq 6000 platform (PersonalBio, Shanghai, China).