title: GSM5911506: D30_WT_pooled_SBNX [H9_Triple-i_Day 30]; Homo sapiens; RNA-Seq
library_construction_protocol: For scRNA-Seq, day 50 organoids (N=4-6 pooled organoids per a processed sample) derived by Triple-i (from ZIP8K8, ZIP13K5, KUCG2 and FOK1 iPSC cell lines), Dual SMAD-i (from ZIP8K8, ZIP13K5, KUCG2 and FOK1 iPSC cell lines), and Inhibitor-free (from ZIP8K8 and ZIP13K5 cell lines) treatments; day 80 individual organoids derived by Triple-i (ZIP8K8 cell line, N=3 individually processed organoids) and Dual SMAD-i (ZIP8K8 cell line, N=3 individually processed organoids), as well as pooled Triple-i organoids (ZIP13K5 cell line, 1  experiment across N=3 pooled organoids); all were dissociated into single cells using papain dissociation kit (Worthington). Organoids were dissected using needle into small pieces, then incubated with papain and DNase I solution for 35-45 min, and later triturated to single cells. Cell suspension was passed twice through 40-micron filter to obtain single cell suspension. Cells were centrifuged at 300g for 5 min, re-suspended in 0.4% BSA in Dulbecco's Phosphate Buffer Solution (DPBS), counted for viability (>80% were viable) and 1000 cells/ µL were used for generating libraries using 10X Chromium single cell 3' reagent kit V3.1 chemistry. For bulk RNA-Seq, RNA was purified using the miRNeasy™ RNA MiniPrep (Qiagene). For scRNA-Seq, roughly 17,400 single live cells were suspended in 0.4% BSA in DPBS buffer (1000 cells/µL) and subjected for GEM generation and barcoding. Library preparation was performed according to the recommended procedures of the manufacture Chromium Single Cell 3' reagent Kit V3.1 chemistry. In day 50 organoids, 10,000 cells were targeted for capturing and 9 cycles were used for cDNA amplification, while 12 cycles were performed for library formation. For day 80 organoids, 11 cycles are used both for cDNA amplification and library formation. Bulk RNA-Seq libraries were generated using the Illumina TruSeq RNA Library Preparation Kits.