RNA-Seq of Chlamydomonas reinhardtii: CC-125 6h replicate 3
We extracted total RNA using the RNeasy Plant Mini Kit (Qiagen) from three biological replicates each of WRC01 and CC-125 at 3, 6, 9, 12, and 48h post-inoculation. Cells were collected by centrifugation at 17,000 g for 1 min, snap-frozen in liquid N2, and stored at -80 C for up to one week before extraction. Cells were not disrupted as specified in the RNeasy protocol, but rather were thawed and immediately mixed with lysis buffer by pipetting. RNA quality was quantified using an Agilent 2100 Bioanalyzer using the Plant RNA Nano assay. We generated cDNA libraries from 500 ng total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) following the standard protocol, with the following exceptions. cDNA fragments were enriched using 14 PCR cycles, based on the results of qPCR using the DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific). Clusters and 100 b.p. paired-end reads were generated on an Illumina Hi-Seq 2500 at the Georgia Institute of Technology. The RNA-Seq experiment was blocked by flow cell, such that each of the three Hi-Seq flow cells contained one replicate of each strain at each time point.