GSM1419592: SMah37Z1.5Ks; Chlamydomonas reinhardtii; RNA-Seq
A volume of culture containing 5 x 10^7 cells was transferred to a 15 ml Falcon tube and centrifuged at 2500 xg for 2 min at room temperature. The supernatant was immediately decanted and the pellets resuspended in 1 ml freshly made lysis buffer (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 20 mM EDTA, 2% sodium dodecyl sulfate). Samples were centrifuged at 600 xg for 3 min at room temperature to pellet starch. The resulting supernatant was snap-frozen in liquid N2 and stored at -80°C for later processing. Upon thawing at room temperature, 10 ml TRIzol (Life Technologies, Carlsbad, California) was added, and the lysates were thoroughly mixed. Samples were incubated for 5 min at room temperature before being transferred to MaXtract HD (QIAgen, Hilden, Germany) tubes. 2 mL of chloroform was added and mixed again. After a 5 min incubation at room temperature, MaXtract HD tubes were centrifuged at 1500 xg for 5 min at room temperature, and the nucleic acid-containing aqueous phase was collected. To extract RNA, samples were processed using the miRNeasy Mini Kit (QIAgen, Hilden, Germany) according to manufacturer’s instructions. To remove contaminating DNA, samples were digested on-column using the RNase-Free DNase Set (QIAgen, Hilden, Germany) according to manufacturer’s instructions. RNA samples were subjected to an ethanol precipitation according to standard laboratory protocol (300 mM sodium acetate and 2.5 volumes of 100 % ethanol) to remove carry-over contaminants from the QIAgen buffers before being resuspended in 50 µL dH2O. We used the Illumina TruSeq Stranded mRNA Sample Prep LS kit according to the manufacturer's instructions (Protocol Rev. D from SEP-2012).
GEO Accession
GSM1419592