HUVEC were collected 6 hrs after seeding onto a collagen matrix and allowed to invade in the presence of S1P, VEGF and bFGF.  Total samples were processed by removing growth media and plucking gels into a M199/Collagenase (1mg/ml) buffer at 37°C for 5 min to obtain a mix containing non-invading and invading cells.   Non-invading samples were collected by removing growth media, rinsinsing with 1X Hepes-Saline +5 mM EDTA and collecting cells on the surface of the collagen (but not invading) with 0.25 %trypsin. Plates were tapped to dislodge cells and the trypsin was neutralized with an equal amoun of serum. Liberated cells were collected and wells were rinsed again with 1X Hepes-Saline +5 mM EDTA to collect remaining non-invading cells. After collection of the non-invading cells, invading cells were collected from the same gels by placing them in a M199/Collagenase (1mg/ml) buffer at 37°C for 5 minfor digestion. All samples were centrifuged at 500 x g for 5 min, supernatant was aspirated,and cell pellets were resuspended in 0.1%BSA in 1X PBS. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GEMs were generated with a Chromium Chip by combining barcoded gel beads, cell suspensions in a master mix, and partitioning oil. To make barcoded, full-length cDNA from poly-A-RNA, GEMs, containing single cells, were  reverse transcribed with an Ilumina TruSeq Read 1 primer, 16 nt 10X barcode, 12 nt UMI and 30 nt poly(dT) sequence. cDNA was purified, amplified and size selected before library construction. Using End Repair, A-tailing, Adaptor Ligation and PCR, libraries were generated with paired-end consructs containing the P5 and P7 Illumina primers.