title: GSM2611744: Chlorella sorokiniana UTEX 1602 grown at pH 5.7 in a modified freshwater medium Sor2; Chlorella sorokiniana; RNA-Seq
library_construction_protocol: M. conductrix and C. sorokiniana UTEX 1602 cells were collected and frozen in liquid nitrogen for total RNA preparation. Briefly, cells were harvested by centrifugation at 14000 g for 1 minute and cell pellets were frozen in liquid nitrogen then stored at -80˚C. Frozen pellets were suspended in 1 mL Trizol and transferred to a bashing beads tube (ZR Fungal/Bacterial DNA Microprep kit, Zymo Research, Irvine, CA) and the solution was vortexed with the beads for 2 minutes, then incubated at room temperature for 5 minutes. Next, 200 µL of chloroform was added to the mixture, vortexed well and centrifuged (12000 g for 3 minutes). The upper layer was carefully collected in an RNase-free tube and mixed with an equivalent volume of 95% ethanol (Sigma) and vortexed well. The solution was further purified using the Direct-zol RNA kit (Zymo Research, Irvine, CA) as directed by the manufacturer, and the total RNA was collected and treated with RNase free DNase I (Zymo Research, Irvine, CA) for 10 minutes and then purified again following the Direct-zol RNA kit. Total RNA quantity and quality were measured using the Nanodrop 2000 spectrophotometer. Poly(A)+ tag based mRNA isolation and RNA sequencing were performed at the University of Minnesota Genomics Center. Poly(A)+ RNA was isolated using oligo(dT) magnetic beads and isolated mRNA was fragmented into small pieces at elevated temperature. The fragmented mRNA was used as template for cDNA synthesis using random primers. Further, 5’and 3’adapters were ligated to the mRNA and the adapter-ligated RNA was reverse-transcribed and amplified using PCR. The final PCR products were size fractioned and used for cDNA sequencing library construction. The cDNA sequencing libraries were sequenced using the HiSeq 2500 Illumina sequencing technique.