Smarter mrna amplification kit

RNA was extracted from the cells following the TRIzol LS protocol guidelines, until the isoproponal precipitation step, then RNA was re-suspended in extraction buffer of a RNA isolation kit (PicoPure, Arcturus), and isolation continued according to the manufacturer’s guideline. This two-step purification protocol helps obtaining RNA of high quality when starting with samples of large volumes, and resulted in a yield of around 8−24 ng per replica with RIN ≥ 8, as measured by a Bioanalyzer (Agilent). Specificity of the RNA was determined by using ~7% of purified RNA for first strand cDNA synthesis with SuperScript III kit (Invitrogen), followed by qPCR (LightCycler 480, Roche) analysis, using lmn-1 as a control to standardized the samples, and hlh-17 to measure differential expression between samples derived from the CEPsh glia to the control. All subsequent steps were performed by the Rockefeller University Genomics Resource Center. Briefly, mRNA amplification and cDNA preparation were performed using the SMARTer mRNA amplification kit (Clontech). Labeled samples were sequenced using an Illumina HiSeq 2000 sequencer using either 50- or 100-base read protocols.