Invitrogen ds cdna synthesis kit

Total RNA was isolated from the cell pellet resuspended in TRIzol reagent (Invitrogen, CA) as described previously (Yang et al., 2009b (link)). Briefly, RNase-free DNase I (Ambion, Austin, TX, United States) was used for each total RNA sample to digest residual genomic DNA, total RNA was subsequently purified using the RNeasy Mini Kit (Qiagen, CA). NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States) was used to quantify the total cellular RNA, and Agilent Bioanalyzer (Agilent, CA) was used to assess RNA quality. Purified RNA with high quality was then used as the template to generate ds-cDNA using Invitrogen ds-cDNA synthesis kit (Invitrogen, CA).

The cells were grown to an OD of 0.3–0.4 in CTFUD medium, centrifuged at 4 °C for 5 min, and immediately flash frozen in liquid N₂. Pelleted cells were resuspended in 1.5 mL of TRIzol (Invitrogen, Carlsbad, CA). Glass beads (0.8 g of 0.1 mm Glass beads; BioSpec Products, Bartlesville, OK) were added to the cell suspension and lysed with 3 × 20 s bead beating treatments at 6500 rpm in a Precellys 24 high-throughput tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was purified using an RNeasy kit (Qiagen, Valencia, CA) with DNase I on-column treatment. RNA quantity was determined by NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and RNA quality was assessed with Agilent Bioanalyzer (Agilent Technologies). RNA (10 µg) was used as the template to generate ds-cDNA using Invitrogen ds-cDNA synthesis kit according to the manufacturer’s protocols (Invitrogen).