GSM8781960: D. Flos-aquae, control C; Dolichospermum flos-aquae; RNA-Seq
All samples were then collected on Whatman filter membranes (Cytiva, Marlborough, USA) and resuspended in 5 ml phosphate-buffered saline before addition of 10 ml RNAProtect reagent (Qiagen, Hilden, Germany). After agitating the samples for 5 seconds, they were incubated for 5 minutes, collected by on Whatman filter membranes (Cytiva, Marlborough, USA) and flash frozen in liquid nitrogen for subsequent use. RNA extraction was performed with a Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, with modifications to the lysis procedure as follows: Lysozyme was added to 2.5 mg / ml and supplemented with 40 µM Spermine as well as 0,2% of the detergent C7Bz0 and 2% of the detergent SB3-14, in addition to 50 mM dithiothreitol (Mehta et al., 2015). Cells were then subjected to homogenization in a Bead Mill Homogenizer (VWR, Radnor,PA, USA) for 2 cycles with 2.4 mm metal beads (VWR, Radnor,PA, USA) and 3 cycles with 0.5 mm glass beads (VWR, Radnor,PA, USA) for 45 seconds each while resting on ice in between steps. RNA samples were quantified using a Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with a 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion sequencing library was prepared by using three probes from QIAGEN FastSelect rRNA 5S/16S/23S Kit (Qiagen, Hilden, Germany), respectively. RNA sequencing library preparation was performed using the NEBNext Ultra II RNA Library Preparation Kit for Illumina, following the manufacturer's recommendations (NEB, Ipswich, MA, USA). In brief, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at the 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using the Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered onto 1 lane of a flowcell. After clustering, the flowcell was loaded onto the Illumina HiSeq instrument according to the manufacturer's instructions. The samples were sequenced using a 2x150bp Paired End (PE) configuration.