3D Culture of SI-NET PDOTS for RNA-seq

RNA-seq was performed as previously described^{1 (link)}. For RNA-seq studies, SI-NET PDOTS were cultured in 3D cell culture chips (AIM BIOTECH). In brief, RNA lysates were prepared from SI-NET PDOTS on Day 9 using the lysis buffer from Agencourt RNAdvance kit (using 1:20 proteinase K). Conditioned media was removed (as described above), before 200 mL of lysis buffer (with proteinase K) was added to each microfluidic chamber. Devices were incubated for 25 min at 37°C, lysates were collected from each microfluidic chamber, and were transferred to RNase-free microcentrifuge tubes, and then stored at −80°C. RNA were extracted using RNAdvance Tissue kit (Beckman Coulter, Cat. No. A32649). RNA quantity and quality were assessed using Quant-iT™ RiboGreen™ RNA Assay Kit (Thermo Fisher, Cat. No. R11490) and Agilent Bioanalyzer RNA 6000 pico kit (Agilent, Cat. No. 5067–1513). RNA libraries were prepared from 10ng RNA per sample using Illumina Truseq RNA Access protocol (Illumina, Cat No. RS-301–2001). RNA-seq was performed at the DFCI Molecular Biology Core Facilities (Illumina NextSeq 500). RNA-seq data were aligned and differential expression analysis were performed using VIPER pipleline, as described^{19 (link)}. CIBERSORT was performed as described^{20 (link)} (https://cibersort.stanford.edu).