nucleic acid sequencing protocol
Affinity chromatography is used to pulldown the protein of interest with attached mRNAs. The protein of interest has a protein A tag encoded on the genome of the yeast strain and IgG (the same antibody is used across all samples) magnetic beads are used to pull out this protein from whole cell extracts. The RNA is processed from these samples and compared to total RNA samples from the same strains.
nucleic acid extraction protocol
samples ground under liquid nitrogen in lysis buffer using a 6870 Freezer Mill (Spex). Lysate thawed on ice, clarified by centrifugation and nucleic acids extract using Trizol Reagent.
nucleic acid library construction protocol
rRNA was depleted from samples using Ribominus Eukaryote Kit, ethanol precipitated and resuspended in DEPC water. Sequencing libraries were then generated using the whole Transcriptome Library Preparation protocol provided with the SOLiD Total RNA-Seq Kit. . cDNA was then amplified and barcoded with SOLiD, RNA barcoding Kit and samples purified using PureLink, PCR Micro Kit
Grown at 30C overnight to an OD600 0.6, pelleted by centrifugation and snap frozen into liquid nitrogen