NextSeq 500 paired end sequencing; High-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton
High-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton
Sample for bulk RNA was collected by filtering 10 ml of culture in exponential phase (density of cells ≈ 10 mil) on 5 µm polycarbonate filters (Whatman).Filters were placed in cryoprotectant tubes with beads (Zirconium oxide beads, Precellys), amended with 600 µl of RLT buffer (Qiagen) which contained 1% of 2-Mercaptoethanol, shock frozen in liquid nitrogen and stored at -80 °C until further processing.  For each cell type we collected samples for bulk RNA. Cells were harvested on 5 µm polycarbonate filters (Whatman). Filters were placed in cryoprotectant tubes with beads (Zirconium oxide beads, Precellys), amended with 600 µl of RLT buffer (Qiagen) which contained 1% of 2-Mercaptoethanol, shock frozen in liquid nitrogen and stored at -80 °C until further processing. Prior to RNA extraction, samples were taken out of -80, thawed on ice and subjected to bead beating (1x 6000 rmp, Precellys Evolution homogenizer). P. tricornutum and Heterocapsa sp. samples were treated for 2 min and T. thermophila just vortexed vigorously. RNA from samples was further extracted with the RNeasy Qiagen kit (QIAGEN, Inc., Valencia, CA) following the kit instructions. RNA was re-eluted in 30 µl of nuclease free water. Libraries were prepared with Magnatrix 8000+ (Nordiag), an eight-channel robotic workstation capable of running custom made scripts for in-tip magnetic bead separations41. Released mRNA–cDNA hybrids were transferred to the robotic workstation where they underwent second strand synthesis, end repair and in vitro transcription. Following transcription, sequencing adaptors are ligated to the RNA, and another round of cDNA synthesis is performed. Each step in the process is followed by a reaction clean-up step.