title: GSM5915334: Monkey NP4; Macaca fascicularis; RNA-Seq
library_construction_protocol: AC, pre-implant hiPS-Cart, and post-implant hiPS-Cart were minced into 1-2 mm pieces. Then the pieces and NP were digested with Liberase solution (for NP, AC, and post-implant hiPS-Cart: RPMI-1640 (Nacalai Tesque) supplemented with 0.2% FBS, 10 mM HEPES pH7.2-7.4, 0.2-0.4 mg/mL Liberase TM (Roche), and 2 kU/mL DNase I (Merck); for pre-implant hiPS-Cart: DMEM with 1% FBS, 1% ITS-X, 50 µg/mL ascorbic acid, 1 mM sodium pyruvate, 1% nonessential amino acids, 1% penicillin streptomycin, 10 ng/mL TGF-β1, 10 ng/mL GDF5, and 10 ng/mL BMP2, 0.2 mg/mL Liberase TM (Roche), and 2 kU/mL DNase I (Merck)) at 37 °C, 5% CO2 for 120-210 minutes with sustained shaking. The cells were agitated 10 times with 1000 μL blue tip and then passed through a 70 μm cell strainer (BD Biosciences), centrifuged at 4°C for 300×g for 5 minutes, and their supernatant was discarded. The cells were resuspended in RPMI-1640 supplemented with 0.2% FBS and 10 mM HEPES. For cell hashing, we biotinylated cell surface proteins with EZ-Link Sulfo-NHS-Biotin (Thermo Scientific), followed by staining with 0.6 μg/mL of Totalseq (A0951-A0955, and A0436 (BioLegend)) (Details of the method will be described elsewhere.) Cells that were alive and stained with A0951-A0955 were selected by flow cytometry using a FACS Aria II flow cytometer (BD Biosciences) and suspended in Sample Buffer (BD Biosciences). TAS-Seq libraries were generated by Immunogeneteqs Inc. (Noda city, Chiba, Japan) according to a previous report: S. Shichino, S. Ueha, S. Hashimoto, T. Ogawa, H. Aoki, W. Bin, . . . K. Matsushima, TAS-Seq: a robust and sensitive amplification method for beads-based scRNA-seq, bioRxiv  (2021). BD Rhapsody scRNA-seq