title: Single-cell RNA-sequencing of mouse embryonic fibroblasts, ESCs, and control and Hic2 overexpressing intermediate reprogramming cells
design: Single-cell RNA-sequencing of mouse embryonic fibroblasts, ESCs, and control and Hic2 overexpressing intermediate reprogramming cells
library_construction_protocol: Nanog-GFP MEFs were reprogrammed using the piggyBac MKOS reprogramming system with overexpression of genes of interest (BFP as a control, or Hic2) described earlier. Cells were collected on days 2, 4, 6 and 9, alongside Nanog-GFP MEFs and Nanog-GFP ESCs. Cells were labelled with a unique Cell Multiplexing Oligo (CMO) provided in the 10x Genomics 3' CellPlex Kit according to the manufacturer's instructions. Subsequently, cells were sorted with the FACS AriaII (BD Biosciences) for either mOrange (reporting for MKOS expression), GFP (reporting for NANOG expression, or both mOrange and BFP. After confirming the viability of cells after sorting using trypan blue and a haemocytometer, 10000 single cells per every 6 samples were pooled together, generating 2 pools that were each processed through the Chromium Single Cell Platform using a Chromium Next GEM Single Cell 3′ GEM Library and Gel Bead kit (v.3.1 chemistry, 10x Genomics) and a Chromium Next GEM Chip G kit following the manufacturer's instructions. After confirming the viability of cells after sorting using trypan blue and a haemocytometer, 10000 single cells per every 6 samples were pooled together, generating 2 pools that were each processed through the Chromium Single Cell Platform using a Chromium Next GEM Single Cell 3′ GEM Library and Gel Bead kit (v.3.1 chemistry, 10x Genomics) and a Chromium Next GEM Chip G kit following the manufacturer's instructions.