GSM1542220: WTrep1_hr09; Dictyostelium discoideum; RNA-Seq
RNA-seq: At each time point, we suspended the cells (5x107cells) with 1 mL of Trizol® (life technologies, CA, USA) and extracted total RNA according to the manufacturer’s recommended protocol. We purified mRNA by two rounds of poly-A selection and fragmented 100 ng of the resulting mRNA into approximately 200-bases fragments. We prepared cDNA and the second strand. We prepared Illumina multiplexed libraries. We prepared 15 (or 20) individual libraries separately (one from each sample) and added a unique barcode to each library at the final step of PCR amplification. We pooled equal amounts of DNA from each library and sequenced one pool per lane of a flow cell on Illumina Genome Analyzer II using the manufacturer's recommended pipeline (versions 1.2 and 1.3, read length = 50 bases)