GSM7133585: Retap rep 1 time 4; Caulobacter vibrioides; RNA-Seq
The cells were pelleted at 20,000 x g for 1 min in 1.5 mL eppendorf tubes to prepare for RNA extraction.Pelleted cells were resuspended in 1mL of pre-warmed (to 65 0C) TRizol Reagent (Ambion) and incubated for 10 min at 65 0C in a heat block. 200 mL of chloroform were added to the samples and tubes were gently inverted a few times. Then samples were incubated at room temperature for 5 min and spun at 20,000 x g for 10 min. The supernatant was moved to siliconized tubes and 500 mL of chloroform was added, vortexed, and spun at 20,000 x g for 10 minutes. RNA samples were precipitated using isopropanol (1x volume isopropanol, 0.1X volume 5M NaOAc pH 5.2) overnight at – 80 0C. The RNA samples were spun at 20,000 x g at 4 0C for 1 hour. Pellets were washed with cold 80% ethanol and spun for 10 min at 20,000 x g, air-dried, and resuspended in 10 mM Tris-HCl (pH 7.0). The RNA-Seq libraries were made using 5µg of total RNA of each sample. Ribosomal RNAs were removed by ribozero gram negative kit and library construction was performed according to the protocol in (Aretakis et al., 2018). mRNA fragments were ligated to 5' adenylated DNA oligo. After reverse transcription, the single stranded DNA was circularized, and PCR amplified [Oh et al,. Cell 147, 1295 (2011)].  Updated adapter oligos that are compatible with the novaseq were used from [Aretakis et al. Methods in Enzymology (2019)].