Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Sample dissociation and FACS were performed essentially as described (Christiaen et al., 2009c; Razy-Krajka et al., 2014). Embryos and larvae were harvested at 12, 14, 16, 18 and 20hpf in 5ml borosilicate glass tubes (Fisher Scientific, Waltham, MA. Cat.No. 14-961-26) and washed with 2ml calcium- and magnesium-free artificial seawater (CMF-ASW: 449 mM NaCl, 33 mM Na2SO4, 9 mM KCl, 2.15 mM NaHCO3, 10 mM Tris-Cl pH 8.2, 2.5 mM EGTA). Embryos and larvae were dissociated in 2ml 0.2% trypsin (w/v, Sigma, T- 4799) ASW by pipetting with glass Pasteur pipettes. The dissociation was stopped by adding 2ml filtered ice cold 0.05% BSA CMF-ASW. The dissociated cells were passed through 40µm cell-strainer and collected in 5ml polystyrene round-bottom tube (Corning Life Sciences, Oneonta, New York. REF 352235). Cells were collected by centrifugation at 800g for 3 min at 4°C, followed by two rounds of washing with ice cold 0.05% BSA CMF-ASW. Cell suspensions were filtered again through a 40µm cell-strainer and stored on ice. Following dissociation, cell suspensions were used for sorting within 1 hour. The B7.5 lineage cells were labeled by Mesp>tagRFP reporter. Contaminating B-line mesenchyme cells were counter-selected using MyoD905>EGFP as described (Christiaen et al., 2008; Razy-Krajka et al., 2014). The TVC-specific Hand-r>tagBFP reporter was used in a 3-color FACS scheme for positive co-selection of TVC-derived cells, in order to minimize the effects of mosaicism. Dissociated cell were loaded in the BD FACS AriaTM cell sorter. 488 nm laser, FITC filter was used for EGFP; 407 nm laser, 561 nm laser, DsRed filter was used for tagRFP and Pacific BlueTM filter was used for tagBFP. The nozzle size was 100 µm. tagRFP +, tagBFP + and EGFP – cells were collected for downstream RNA sequencing analysis. Reverse transcription and cDNA amplification were carried out using modified Smart-seq2 protocol (Picelli et al., 2013). Single cells were sorted by FACS as described above into 96-well plates and collected in 3.4 µl RT buffer (0.5 µl 10µM 3’ RT Primer ( 5' - AAG CAG TGG TAT CAA CGC AGA GTA C T30 VN - 3’), 0.5µl 10µM dNTP Mix, 0.5 µl 4U/µl RNase Inhibitor, 1 µl Maxima RT Buffer, 0,9 µl nuclease-free water) in each well. Plates were stored at -80°C or processed immediately. Plates were incubated at 72°C for 3min and chilled on the ice to denature the template RNA. 2µl RT reaction mixture (0.5µl 10um TSO primer (5′-AGACGTGTGCTCTTCCGATCTNNNNNrGrGrG-3′), 0.925µl 5M Betaine, 0.4 100mM MgCl2, 0.125µl 40U/ RNAase inhibitor, 0.05µl 200 U/µl Maxima H Minus Reverse Transcriptase) were added to each well. Reverse transcription was carried out by incubating the plate at 42°C for 90 min, followed by 10 cycles of (50 °C for 2 min, 42 °C for 2 min) and heat inactivation at 70° for 15 min. 7µl PCR amplification mixture (0.25µl 10 μM PCR primer (5′AGACGTGTGCTCTTCCGATCT-3′), 6.25µl KAPA HIFI ReadyMix, 0.5µl nuclease-free water) were added to each well. PCR amplification was carried out with a denaturation at 98 °C for 3 min, followed by 21 cycles of (98 °C for 15 s, 67 °C for 20 s, and 72 °C for 6 min) and a final extension at 72° for 5 min. PCR products were purified by adding 10µl (0.8×) Agencourt AMPureXP SPRI beads (Beckman-Coulter) to each well, followed by 5min incubation and two rounds of wash using 100µl freshly prepared 70% Ethanol at room temperature. Purified cDNA were eluted in 20µl TE. The concentration of amplified cDNA was measured across the entire plate using Picogreen assay. The concentration of amplified cDNA was in a 0.5–2 ng/μl range. Fragment size distribution was checked for randomly selected wells with High-Sensitivity Bioanalyzer Chip (Agilent), the expected size average should be ~2 kb. For each sample, the amplified cDNA were normalized to a working concentration ranging from 0.1 to 0.2 ng/µl with TE buffer. 1.25 µl of diluted cDNA from each well were used for library preparation. Single cell libraries were prepared using the Nextera XT DNA Sample Kit (Illumina) according to manufacturer's instructions. After library amplification, 2.5μl from each well were pooled into a single 1.5-ml microcentrifuge tube, purified using Agencourt AMPure XP beads and eluted with 30μl TE buffer. 1μl purified library was used to measure the fragment size distribution using the Agilent HS DNA BioAnalyzer chip and another 1μl of the purified library was loaded into Qubit fluorometer to estimate library concentration according to the manufacturer's instructions. Libraries were sequenced on an Illumina HiSeq 2500 sequencer to obtain paired-end 25bp reads.