GSM8911852: run1_Day0; Homo sapiens; RNA-Seq
For single cell library prep, ~30 3D cysts structures for each time point were collected and dissociated using Trypsin/EDTA for 5 min at room temperature. Cells were then washed with PBS, and the resulting cell suspension was used to sort individual live cells in 96 well plate. Full length single cell RNA-seq was performed using a modified version of the Smart-seq2 protocol. Briefly, individual cells were sorted into 96 well plates containing lysis buffer in presence of RNase inhibitor, dNTPs and OligodT. Reverse transcription of the polyadenylated RNA was performed with Maxima H Minus Reverse Transcriptase and Template Switching Oligos. The resulting cDNA was amplified with 23 cycles of PCR and libraries were prepared for sequencing with miniaturized NexteraXT Illumina protocol. Libraries were sequenced on Illumina NextSeq 500 System (single-end 75bp reads).