TITLE: Cell sorting of marine heterotrophic flagellates for single-cell genome amplification
AUTHORS: Camille Poirier, Raquel Rodríguez‐Martínez, Emily Cook, David S. Milner, Alexandra Z. Worden, Thomas A. Richards

[DESCRIPTION]
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[STEPS]
?. [Water collection and preparation]
Gently resuspend the cells from the filter using 1-2 ml sterile artificial sea water (ASW) and stain a 500 µl volume of the cell concentrate with 10 µM Paclitaxel, Oregon Green® 488 Conjugate (to target cytoskeletal tubulin). Incubate for 1 hour at room temperature.
Use a 1:100 dilution of a 1 mM Paclitaxel, Oregon Green stock solution (prepared in DMSO and stored at -20°C).
?. [Water collection and preparation]
Also stain with 2 µg/ml Hoechst 33342 (to target dsDNA) for 30 mins at room temperature.
?. [Flow cytometry and sorting]
Use sort window combinations to select cells that show both green and blue fluorescence (520/35 nm bandpass filter for Oregon Green [tubulin] and a 460/50 nm bandpass filter for Hoechst 33342 [dsDNA]) compared to unstained control samples, and that show baseline red fluorescence (692/40 nm bandpass filter) indicating the absence of chlorophyll. This will allow for the exclusion of the majority of photosynthetic cells.
Regularly check the sort quality by sorting a known number of fluorescent beads onto a slide, and counting them on an epifluorescence microscope. Do not add beads to actual sort samples (to limit possibility for contamination).Please note that this protocol was performed using a BD Influx and it is possible that other cell-sorters would require different validation methods.
?. [Flow cytometry and sorting]
Perform cell sorting on a flow cytometer equipped with 488 nm and 355 nm lasers. Use sterile nuclease-free 1x PBS (pH 7.4) as sheath fluid.
?. [Flow cytometry and sorting]
Prior to sorting, illuminate 96-well plates by UV radiation inside the sort chamber (2 mins).
?. [Flow cytometry and sorting]
Sort targeted cells into 96-well plates (one cell per well; Single-Cell sorting mode in BD FACS ‘Sortware’ software). Leave the outer column of wells empty for sheath-fluid only negative controls.
?. [Flow cytometry and sorting]
Cover the plates with foil and place at -80°C immediately after the sort.
?. [DNA amplification for single cell genome or amplicon sequencing]
UV-treat all materials (except cell samples) in a HL-2000 HybriLinker (UVP) for 30-90 mins prior to conducting multiple displacement amplification (MDA).
?. [DNA amplification for single cell genome or amplicon sequencing]
Lyse samples (both sorted cells and negative controls) for 10 mins at 65°C using buffer D2 from the REPLI-g Single Cell Kit (Qiagen), according to the manufacturer's instructions for amplification of genomic DNA from single cells.
?. [DNA amplification for single cell genome or amplicon sequencing]
After neutralization, amplify samples using the REPLI-g kit in a final volume of 50 µl. Perform MDA reactions in a thermal cycler for 8 h at 30°C with the lid temperature set to 65-70°C. After amplification, aliquot samples as follows:a) 2 µl diluted 100-fold for PCRs (see step 14; stored at -20°C)b) 20 µl kept as a backup (stored at -80°C)c) 28 µl (stored at -80ºC). Use 25 µl of this aliquot for sequencing (step 17)
?. [Screening MDA products by amplification of the V9 region of the nSSU rRNA gene]
Dilute an aliquot (2 µl) of each MDA product 100-fold in nuclease-free water and use 2-5 µl of this dilution as the template for 25 µl PCR reactions. Carry out PCR amplification using the primers:1389F (5’-TTGTACACACCGCCC-3’)1510R (5’-CCTTCYGCAGGTTCACCTAC-3’)Use Phusion High-Fidelity DNA Polymerase (NEB) in a 25 μl reaction volume, with 0.35 μM final concentration of each primer, 3% DMSO and 2X GC buffer.Initial denaturation step of 98°C for 30 sFollowed by 25 cycles of:98°C for 10 s57°C for 30 s72°C for 30sThen a final elongation step of 72°C for 10 mins
Primers from Amaral-Zettler et al. (2009); PCR cycling conditions from Logares et al. (2014).
?. [Screening MDA products by amplification of the V9 region of the nSSU rRNA gene]
Run PCR products on a 1% agarose gel stained with GelGreen. Excise bands using a blue light transilluminator to prevent DNA damage.
?. [Screening MDA products by amplification of the V9 region of the nSSU rRNA gene]
Purify amplicons using a GeneJet gel extraction kit (Thermo Scientific), quantify with a Qubit fluorometer using the dsDNA BR kit (Invitrogen) and send for Sanger sequencing (Eurofins Genomics).
?. [Library preparation]
Purify an aliquot (25 µl) of each chosen MDA sample (including 6 negative controls: 4 negative wells from sorting plate, plus 2 buffer controls from the kit) using AMPure XP beads following the manufacturer's instructions.
?. [Library preparation]
Quantify using a Qubit fluorometer and dilute in 10mM TrisCl (pH 8.0) to 1 µg in a final volume of 130 µl. For the negative controls add the total amount of sample (~20µl) plus TrisCl until 130 µL (~110µL).
?. [Library preparation]
Denature library pools, dilute and perform 250 bp paired-end sequencing across two lanes on a HiSeq 2500 using Rapid Run SBS v2 reagents (Illumina).
?. [Library preparation]
Prepare libraries using a Nextflex Rapid DNA library preparation kit and indexes (BIOO Scientific) without PCR amplification.
Method i. Pool 3 µl of each sample and concentrate, selecting 450-650 bp products using a Blue Pippin 1.5% agarose cassette (R2 marker). Average size of the recovered libraries is ~420 bp (with 295 bp inserts).
Method ii. Prepare libraries using bead-based size selection (420-620 bp), quantify by qPCR and pool equimolar amounts (2 nM).
?. [Water collection and preparation]
Pre-filter the water sample through a 30 µm mesh, then concentrate by gravity filtration ~100x onto a 0.8 µm filter to approximately a 1-2 ml volume.
?. [Water collection and preparation]
Pellet cells by centrifugation (3 mins at 6,000 x g) and wash with 2 ml ASW to remove unbound dye. Repeat centrifugation and resuspend in 2 ml ASW.
?. [DNA amplification for single cell genome or amplicon sequencing]
Negative controls include:4 cell-sort controls per 96-well plate (outer wells)2 buffer-only controls from the MDA kit (i.e. no sample added)
?. [Library preparation]
Fragment DNA using focused acoustic waves (Covaris E220) and then concentrate the sample.