GSM5870280
The SV and BA were dissected from using fine forceps and pooled in a Tyrode's solution (pH 7.4) following washing in 1X PBS. The pooled tissue was washed 3 times in Tyrode's before the addition of Liberase IV (0.29U/ml, Roche, 05401020001), Elastase (1.0 U/ml, Sigma, E7885), and Pronase E (0.92 U/ml, Sigma, P5147). Dissociation of the tissues was facilitated via incubation at 37°C and gentle trituration with pipettes of descending tip diameter (first 2.0 mm followed by 0.8mm. Can also use filtered P1000 and P20 tips, respectively). The dissociation reaction was halted by transferring the cells to a modified Kraft-Brühe solution (pH 6.9) and the cells were spun down to a pellet before being resuspended in a final 1% BSA solution in 1X PBS. The 10x Genomics Chromium 3' v2 assay standard protocol was followed as previously described. Sequencing was performed on the Illumina HiSeq-4000 platform at a target depth of 230 million reads per simple.