title: Metatranscriptomic Sequencing of Anderson Lake: Isolated on 2022-05-02: replicate 1
design: We performed RNA sequencing on five dates total, one in mid-April (April 18th) and four dates across May (May 2nd, May 9th, May 16th, and May 23rd). Our April sample captures pre-bloom conditions, while the dates across May were designated as early, mid-early, mid-late, and late bloom. For each date we included a biological replicate (10 samples total). Each sample was filtered with a 0.40 m pore-size EMD Millipore Durapore Membrane Filter (Millipore, Foster City, CA, USA). The filter with concentrated biomass was aseptically inserted into a Lysing Matrix A bead tube (MP Biomedicals, Santa Ana, CA, USA) containing 600 L of RLT plus buffer (QIAGEN, Valencia, CA, USA) and stored at -80 C before use. For DNA/RNA extraction, the filter was disrupted and lysed for 1 min using a Mini-Beadbeater-16 (BioSpec Products, Inc., Bartlesville, OK). The mixture was then centrifuged at 12,000  g for 3 min at room temperature. The lysate was moved to a DNeasy column, and the DNA/RNA was extracted using the AllPrep Mini Kit following the manufactures protocol. RNA samples were further purified using TURBO DNase (Invitrogen, ThermoFisher Scientific, Waltham, WA, USA) to remove genomic DNA following manufacturers protocols. Total RNA quality and quantity were assessed using a BioAnalyzer 2100 (Agilent, Santa Clara, California, USA) and determined to be of good quality with RIN values ranging from 5.4-8.3. Additionally, RNA concentrations were measured with both a Qubit 2.0 (ThermoFisher Scientific, Waltham, MA, USA) and a NanoDrop spectrometer (ThermoFisher Scientific, Waltham, MA, USA) and samples were diluted to a starting amount of 100 ng of total RNA in 11 L volume of molecular grade water.  In all, 10 sequencing libraries (a single pre-bloom control dates and four experimental bloom dates spanning from early to late (maximum) bloom) were constructed using Illumina Stranded Total RNA Prep with Ribo-Zero Plus (Illumina, San Diego, CA, USA) following manufacturers instructions and reaching the final expected size of 330-430 bp (average ~382bp). Individual libraries were pooled in equimolar concentrations and the final pooled libraries were quantified using KAPA library quantification kit for Illumina (Roche, Basel, Switzerland). The pooled libraries were diluted to a concentration of 4 nM, and then denatured and diluted to 1 pM according to the manufacturer's instructions. For each sample condition all libraries were indexed and sequenced on a single flow cell on a Nextseq550 platform using the Nextseq550 High Output Kit v2.5 (Illumina, San Diego, CA, USA)