GSM8458083: plankton_J7_S65; marine plankton environmental sample; RNA-Seq
Cells were captured using the CellCelector platform (ALS Automation GmbH/Sartorius) with a glass capillary tube (inner diameter:  150 μm) using the manual picking mode. The cells were dispensed into  a 384-well Frame-Star plate (4titude Ltd, Surrey, UK) containing 1 μL lysis buffer, which was cooled to  4 ºC  for the duration of the capture. For the first batch of samples, a brightfield image of each cell was recorded before capture. For the second batch of samples, brightfield and epifluorescence images (emission spectrums 515, 595 and 681 nm) of each cell were taken before each capture. For both batches, a brightfield image was taken after each cell capture to confirm successful picking. Two of the wells included in the following cDNA synthesis, library preparation and sequencing did not have cells placed into them, to act as a control for background contamination originating from the laboratory equipment used. Following cell capture, the lysis plate was stored at –80 C for one month cDNA synthesis was performed following the Smart-seq2 protocol [29]. In brief, 1.8μl of reaction mix (SmartScribe, Rnase Inhibitor, 5x First Strand Buffer, DDT (100mM), Betaine (5M), MgCl2 (1M), TSO (100uM), H2O) was added to each well containing a single cell lysate using the MANTIS automated liquid handler and reverse transcription was performed by incubating the plate at 42 C for 90 minutes, then 70 C for 5 minutes using a thermal cycler (check brand). Following this, 4.2μl of PCR mix (H2O, Kapa Buffer (5x), dNTP (10mM), Polymerase (1U/ul), IS_PCR (10uM)) was added to each well using the MANTIS automated liquid handler (brand) and PCR was performed according to the following cycle: 1) 95 C for 3 minutes, 2) 26 cycles of 98 C for 20 seconds, 67 C for 15 seconds, 72 C for 5 minutes, and 3) 72 C for 5 minutes. A mean concentration of the cDNA in each well was calculated using measurements from the Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) and cDNA concentrations were normalised to 1.4 ng/µl with nuclease-free water using the automated liquid handler Mosquito. The plate was stored at 4C until library preparation. The Nextera XT kit (Illumina, San Diego, CA, USA) was used for tagmentation using 15 cycles of PCR. Bead purification was carried out using Agencourt Ampure XP (Beckman Coulter, Brea CA, USA) magnetic beads. One sample was purified at a ratio of 0.7X for two cycles and a second sample was purified using an additional reverse purification at a ratio of 0.5X for one cycle. Library concentration and size were compared between the two samples using the High-Sensitivity D1K TapeStation (Agilent 2200 TapeStation).