title: 16S rRNA Seq of Chlorella mixed culture: Chlorella strain C596 cultures grown from the as-recieved culture R1 replicate 3
design: Total RNA was extracted and purified using the RNeasy Plant Mini Kit (Qiagen)(Mansfeldt et al., 2016) and its integrity and concentration were evaluated using a RNA 6000 Nano Kit on an Agilent Bioanalyzer 2100 (Agilent Technologies).  Complimentary DNA (cDNA) was generated using an iScript reverse transcriptase and a blend of olig(dt) and random hexamer primers (iScript cDNA Synthesis Kit; Bio-Rad).  The 16S rRNA gene was amplified using the universal bacterial primers 341F (5’- CCTACGGGNGGCWGCAG -3’) and 805R (5’- GACTACHVGGGTATCTAATCC -3’)(Herlemann et al., 2011), with overhang sequences compatible for index attachment as described elsewhere (Bell et al., 2016).  PRIME HotMasterMix (5 PRIME Inc., Gaithersburg, MD, USA) was used in PCR reactions, and cycle conditions were set as previously published (Howard et al., 2017).  The 16S gene amplicon pool was sequenced on an Illumina MiSeq (Cornell Genomics Facility, Ithaca, NY) using a 600-cycle MiSeq Reagent Kit v.3.  Sequences were processed using the Brazilian Microbiome Project pipeline with modifications as described by Howard et al., 2017.