GSM7518374: Ctrl_rep5; Mus musculus; RNA-Seq
The organoids were harvested on day 12 of culture from Matrigel droplets by using ice cold 5mM EDTA-PBS. Replicate droplets were collected, combined and incubated on ice in a large volume of buffer until the Matrigel had dissolved. The solution containing soluble Matrigel and the organoids was centrifuged (100 x g, 5 min, 4C) and the pellet was washed once with ice cold PBS. Total RNA was isolated from cell pellets by using Trisure reagent (Bioline, USA) according to manufacturer's instruction. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers followed by the second strand cDNA synthesis. The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.