title: scRNA-seq of Botrylloides leachii active colony blood cells
design: Whole hemocyte cells were extracted by puncturing marginal ampullae with a needle (27G) according to the established technique (26). The cells of each sample were collected in a 1.5ml tube and the amount of cells was estimated by hemocytometer under the microscope. The number of hemocytes was diluted in different samples in order to start with similar amount of cells.  Assays were prepared following Drop-seq protocol (Bageritz et al., 2019). Single cells (100/l) from hemocyte suspensions of active (n=3) and torpid (n=2) colonies were inserted in a droplet containing barcoded beads (120/l, ChemGenes Corporation, Wilmington, MA) using a microfluidic polydimethylsiloxane device (Nanoshift) at rates of 4000l/h for 15min each colony. Droplet breakage was then performed using perfluoro octanol (Sigma-Aldrich). Beads were harvested, the hybridized mRNA transcripts were reverse transcribed (Maxima RT, Thermo Fisher) and the left primers were digested by exonuclease I (New England Biolabs). Following that, the beads were washed, counted and aliquoted for pre-amplification (2000 beads/reaction, equals ~100 cells/reaction) using Polymerase Chain Reaction (PCR) solution (12 cycles; primer: AAGCAGTGGTATCAACGCAGAGT [100M], 2 KAPA HiFi Hotstart Readymix [KAPA Biosystems]). Cycles conditions: 3min 95C, 4 cycles of 20s 98C, 45s 65C, 3min 72C, followed by 8 cycles of 20s 98C, 20s 67C, 3min 72C, then 5min at 72C. The PCR products of all individual cells from each sample were pooled together and purified (X2) by 0.6 clean-up beads (CleanNA), according to manufacturers instructions. Then, 1ng of pre-amplified cDNA from the estimated 1000 cells in each sample, were tagged by Nextera XT (Illumina) with a custom P5 primer (Integrated DNA Technologies). Single-cell libraries were sequenced in a 100bp paired-end run on the Illumina HiSeq4000 using 0.2nM denatured sample and 5% PhiX spike-in. We used 0.5M Read1CustSeqB (primer sequence: GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) for read 1 priming.