GSM5596834: Day0_undifferentiated_iPSCs_bulk_RNAseq_rep2; Homo sapiens; RNA-Seq
Frozen tissues were carefully transferred to a stack of chilled aluminum foil kept on dry ice to prevent thawing. The folded foil encapsulating the tissues were placed on a block of dry ice and the foil was pounded with a pestle to pulverize the tissues into powder. 1mL of lysis buffer that contains nuclei buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, pH 7.4), 0.1% IGEPAL CA-630, 1% SUPERase In RNase inhibitor (20 U/μL, Thermo), and 1% BSA (20 mg/mL, NEB) were added onto the tissue powder and transferred to a 1.5mL tubes. Samples were incubated in the lysis buffer for 1 hour on ice. The samples were pipetted up and down with pre-cut 1000uL pipette tip to further disassociate the tissue. The dissociated tissues were passed through 70 um cell strainers (Corning) into a 50mL conical tube. The strainers were rinsed with lysis buffer to minimize nuclei loss. The samples were centrifuged to pellet the nuclei at 500g for 5 minutes at 4°C and the supernatant was discarded. The samples resuspended again in 1ml lysis buffer, transferred into new 15mL tubes, pelleted again and supernatant was discarded. The pellets were resuspended in 50ul of nuclei buffer, and 5 mL of 4% Paraformaldehyde (PFA) (EMS) diluted in RNase free PBS, was added to fix the nuclei for 15 minutes on ice. The tubes were flicked gently every 5 minutes to reduce clumping of nuclei. The fixed nuclei were pelleted at 500g for 3 minutes at 4°C and the PFA waste was discarded. The pelleted nuclei washed in nuclei wash buffer (cell lysis buffer without IGEPAL) and then centrifuged again at 500g for 5 minutes 4°C, and the supernatant was discarded. Finally, the pellets were resuspended again in nuclei wash buffer and then flash-frozen in liquid nitrogen before storing in –80°C. For nuclei extraction from the differentiation culture, the cells were treated with StemPro Accutase (Theromo, #A1110501) for 7min to detach the cells and transfer them into 15mL tube, then incubated in trypsin (Theromo, #25300054) for another 7min to prevent re-clumping. The cells were span down to remove trypsin after inactivation with more media. The pellet was treated with nuclei lysis buffer and the same steps for nuclei extraction protocol were followed. Single-cell combinatorial-indexing RNA-sequencing (sci-RNA-seq) protocol is described previously and more details can be found in this link (https://github.com/bbi-lab/). sci-RNA-seq relies on the following steps, (i) thawed nuclei were permeabilized with 0.2% TritonX-100 (Sigma, #T9284) (in nuclei wash buffer) for 3 min on ice, and briefly sonicated to reduce nuclei clumping; (ii) nuclei distributed across 96-well plates; (iii) A first molecular index is introduced to the mRNA of cells within each well, with in situ reverse transcription (RT) incorporating the unique molecular identifiers (UMIs); (iv) All cells were pooled and redistributed to multiple 96-well plates in limiting numbers (e.g., 10 to 100 per well) and a second molecular index is introduced by hairpin ligation;(v) Second strand synthesis, tagmentation, purification and indexed PCR; (vi) Library purification and sequencing is performed.