Transcriptional profiling by single cell-RNA sequencing of HARA cells according to Calbindin and HERVH-LTR7 activity
Transcriptional profiling by single cell-RNA sequencing of HARA cells according to Calbindin and HERVH-LTR7 activity
Calbindin-deficient HARA 3D5 cells were generated by Cas9-mediated mutation of the CALB1 gene, encoding Calbindin. HARA.LTR7-GFP+ and HARA.LTR7-GFP- cells were generated by introduction of a HERVH LTR7-GFP reporter into parental HARA (Calbindin-sufficient) cells, using the Sleeping Beauty transposon system. Cells transduced and expressing the reporter were distinguished from cells either not transduced or not expressing GFP expression. GFP+ and GFP- cells were enriched by cell sorting and kept as separate sublines. Introduction of the HERVH LTR7-GFP reporter does not directly affect Calbindin expression, but GFP reporter expression is a marker for and correlates with Calbindin expression. All four cell populations, were grown in vitro and were processed as one batch at the same time, with cell viability between 89% and 93%. RNA was isolated and reverse transcribed with the 10× 3'-mRNA Seq (v3) kit (10× Genomics). Library prep was performed using the the 10× 3'-mRNA Seq (v3) kit (10× Genomics).