GSM8853876: AGS, siNC, rep2; Homo sapiens; RNA-Seq
Discard culture medium and wash cells twice with PBS. Add 1 mL TRIzol reagent per 6-well plate to lyse cells. Transfer lysate to RNase-free tubes and incubate at room temperature for 5 min. Add 0.2 mL chloroform, vortex for 15 sec, and incubate for 5 min. Centrifuge at 12,000 rpm for 15 min at 4°C. Transfer aqueous phase to a new tube, mix with 0.5 mL isopropanol, and incubate for 10 min. Centrifuge at 12,000 rpm for 10 min at 4°C. Wash RNA pellet with 1 mL 75% ethanol (prepared with DEPC-treated water). Centrifuge at 7,500 rpm for 5 min at 4°C. Air-dry RNA pellet for 5-10 min and dissolve in DEPC-treated water. RNA concentration and purity were measured using Nanodrop, and integrity was assessed via Agilent 2100 Bioanalyzer. 1. mRNA was enriched using poly-A selection with magnetic beads. 2. mRNA was fragmented to 200-300 bp. 3. Double-stranded cDNA was synthesized using reverse transcriptase. 4. End repair and A-tailing were performed. 5. Sequencing adapters were ligated. 6. Libraries were amplified via PCR and validated using Bioanalyzer.