GSM7666412: Set1 3D culture (N/A) and with T cells, 3D triculture (N/A/M), and 3D PiChip (N/A/M/T cells)  in CTRL (G10) and AD (H10); Homo sapiens; RNA-Seq
Cells treated with Accutase at room termperature for 10mins and then dissociated to disperse into single cells. Shorlty after that Live/cell dead assay performed to count live cells and then loaded into InDrop in-house equipment for the capturing of the single cells. After generating barcoded cDNA from single cells, the droplets are broken and cDNA is processed for next-gen sequencing (Nature Protocol, ; doi:10.1038/nprot.2016.154). Briefly, purification of the cDNA library and second-strand synthesis, followed by in vitro transcription (IVT) using T7 RNA polymerase. The amplified antisense RNA is fragmented using zinc-ion-mediated cleavage, and converted into a DNA library ready for Illumina sequencing by a second RT reaction and a few cycles of PCR. This library preparation step takes ~2 d and includes two quality control (QC) steps. As detailed in Figure 3, a library index is added during the final PCR to allow pooling of multiple libraries for sequencing in a single run. Our protocol is designed to work with all Illumina sequencing platforms and can possibly be adapted to work with different sequencers.