GSM8383276: Jejunum, LEC, Sv-2; Mus musculus; RNA-Seq
RNA was harvested from 250 sorted blood and lymphatic endothelial cells using RNA cleanup XP beads RNA was eluted for 1 min in RT-cDNA synthesis mix 1 (0.5 µl P1-T31 (20µM), 0.3 ml RNasinplus (Promega), 1.5µl 10µM dNTP, 3.5 µl 10µM Tris pH 7.5-0.5% IGEPAL CA-630 (Sigma) and 1.7 µl RNase free ddH2O) and pipetted up and down to mix. The eluted sample was then incubated for 3 min at 72ºC, followed by 1 min on ice, then 7.5 µl of mix 2 was added (3µl 5X RT Buffer, 0.375 µl 100 mM DTT, 0.375 µl RNasin plus, 0.5 µl P1-RNA-TSO (40 µM), 0.75 µl Maxima RT Minus H (Thermo Scientific), 1.8 µl 5M Betaine (Sigma), 0.9 µl 50 mM MgCl2 and 0.175 µl RNase free ddH2O and mixed well. Samples were placed in a thermocycler and subjected to the following protocol: 42ºC for 90 s, (50ºC for 2 min, 42ºC for 2 min) x10 cycles, 70ºC for 15 min. The cDNA was then amplified using 13.5 µl cDNA, 20 µl KAPA HiFi 2x Mix, 1.5µl P1-PCR, and 5 µl H2O. Samples were subjected to the following protocol: 98ºC for 3 min, (98ºC for 15 s, 67ºC for 20 s, 72ºC for 6 min) x12 cycles, 72ºC for 5 min. cDNA was cleaned up using RNA Clean XP beads and eluted in water. 0.25ng cDNA was used as input and tagemented using Nextera XT index kit per manufacturers instructions with 0.5X the indicated volume.