Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Cells were trypsinized with TrypLE Select and cell resuspensions were kept in maintenance medium with albumin from bovine serum until loading on the Fluidigm chip. Fluidigm C1 Autoprep System Cells medium (10-17 micrometer) microfluidic was used to capture the cells. 14 microlitre of cell suspension (approx. 800 cells/microlitre) was mixed with 7 microlitre C1 Suspension Reagent after filtering. Single-cells were then captured for 30 min at 4°C using the “Cell Load (1772x/1773x)” script. The protocol for Lysis, RT and PCR was performed as previously described (Islam et al., Nat Methods. 2014 Feb;11(2):163-6). Amplified cDNA was harvested with 13 microlitre Harvest Reagent and cDNA library quality was measured on an Agilent BioAnalyzer. Cell barcoding and fragmentation was performed in a single step using Tn5 DNA transposase as described previously. 1 microlitre Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were resuspended in 20 microlitre Binding and Blocking buffer (10mM Tris, 250mM NaCl, 5mM EDTA, 0.5% SDS) and added to each well. After 15 min incubation at room temperature, all wells were pooled, the beads washed once with 100 microlitre Washing buffer (10mM Tris-150mM NaCl, 0.02% Tween), once in 100 microlitre Qiagen Qiaquick PB and then twice using 100 microlitre Washing buffer. Restriction was performed to cleave 3' fragments: the beads were incubated in 100 microlitre restriction mix (1x NEB CutSmart, 0.4 U/microlitre PvuI-HF enzyme) for 1h at 37°C. Finally, the beads were washed three times with Washing buffer, then resuspended in 30 μl ddH2O and incubated for 10 min at 70°C to elute the DNA. AMPure beads XP (Beckman Coulter) were used at 1.8x volume and eluted in 30 microlitre to remove short fragments.