GSM5695630: Pig ESCs cultured without feeder cells (LDN-treated); Sus scrofa; RNA-Seq
Before RNA and DNA extraction from pig ESCs cultured with feeder cells, SSEA4-positive undifferentiated pig ESCs were sorted using a MACS Cell Separation System (Miltenyi Biotec, Germany) to remove differentiated cells and feeder cells. The dissociated pig ESCs were reacted with an anti-SSEA4 antibody (1:200; MAB4304, Millipore) and anti-mouse IgG microbeads (1:5; Miltenyi Biotec). SSEA4-positive cells were sorted on an LS column (capacity, 1 × 108 magnetically labeled cells; Miltenyi Biotec) according to the manufacturer's instructions. Total RNA was extracted from pig fetal fibroblasts, and SSEA4-positive ESCs were sorted using TRIzol reagent (Invitrogen). cDNA library preparation and RNA-Seq analysis were performed by the National Instrumentation Center for Environmental Management (NICEM) at Seoul National University (www.nicem.snu.ac.kr, Seoul, Korea). The viability and number of cells were calculated by LUNA-FL™ Dual Fluorescence Cell Counter (Logos Biosystems, Anyang, Korea) before 10x Genomics single-cell RNA-seq library construction. The cells were loaded into chromium microfluidic chips using the Chromium Single Cell 3ʼ Reagent Kit (v3 chemistry; 10×Genomics, Pleasanton, CA, USA) and then separated into single-cell gel bead emulsions (GEMs) by the Chromium Controller (10×Genomics) according to the manufacturer's protocol. Reverse transcription of RNA molecules captured from single cells in GEMs was performed by a C1000 Touch Thermal Cycler (Bio–Rad, Hercules, CA, USA). Then, subsequent steps, including cDNA amplification and library construction, were performed according to the manufacturer's instructions. The cDNA libraries were quantified by a LabChip GX Touch Nucleic Acid Analyzer using a DNA High Sensitivity Reagent Kit (PerkinElmer, Waltham, MA, USA).