Agilent bioanalyzer rna 6000 nano kit

RNA was isolated from two biological replicates for each test sample using the hot acidic phenol protocol described in Current Protocols in Molecular Biology (1996) Unit 13.12 [34 ]. Briefly, the cell pellet was washed with nuclease free water once and resuspended thoroughly in TES (10 mM TrisCl pH 7.5, 10 mM EDTA and 0.5% SDS). 400 μl acidic phenol pH 4.5 (0.1 M citrate buffer saturated) was added to the suspension and incubated at 65 °C for 60 min with occasional vortexing. Samples were spun at 14,000 rpm for 10 min at 4 °C. The top aqueous layer was transferred to a fresh tube. Subsequently, 400 μl chloroform was added, mixed, and spun as above. The aqueous layer was taken and mixed with 1/10th volume 3 M NaAc pH 5.2 and 2.5 volumes of 100% ethanol. The samples were precipitated at -80 °C overnight, then spun as above and the pellet was washed with 70% ethanol, dried, and resuspended in 50 μl nuclease free water. RNA quality was checked by 1.2% agarose formamide gel electrophoresis. RNA quality and quantity were also assessed using the Agilent Bioanalyzer RNA 6000 Nano kit (Agilent Technologies) and the Qubit HS RNA kit (Thermo Fisher Scientific). Samples were sent to Clevergene for RNA-seq.

Total RNA from cells was isolated using RNeasy Mini Kit (QIAGEN, USA). RNase-Free DNase treatment was performed on column to remove residual DNA. Twelve RNA samples were assessed for concentration and quality using the Thermo Scientific (Waltham, MA) NanoDrop 8000 Spectrophotometer. RNA sequencing libraries were constructed using Illumina TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA) per manufacturer's instructions. Briefly, total RNA with RNA integrity (RIN) scores of 9 or better using Agilent Bioanalyzer RNA 6000 nano kit were used for RNA library preparation. mRNA was then purified from one microgram of total RNA from each sample using oligo-dT attached magnetic beads with two rounds of purification. Subsequently, the mRNA was fragmented and primed for cDNA synthesis according to manufacturer's recommendations. RNA adapters and barcodes were ligated to cDNA to allow for clonal amplification and multiplexing on a single lane on a single end Illumina flow cell. Sequencing was done on an Illumina HiSeq 2500 to an average read depth of 26 million reads per sample and the minimum number of reads for a sample was at least 23 million reads.