MLL2 facilitates long-range gene regulation through LINE1 elements [scRNA-seq]
MLL2 facilitates long-range gene regulation through LINE1 elements [scRNA-seq]
Cells were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific, 25030024), 0.1 nM β-mercaptoethanol, 1x NEAA, 1x antibiotic and antimycotic solution and 1 µM retinoic acid (RA; Sigma-Aldrich, R2625) for 4 days. Single-cell suspensions were fixed using the Evercode™ Cell Fixation workflow compatible with the Parse Biosciences Evercode WT Mini scRNA-seq kit. Briefly, freshly isolated cells were washed and incubated with the fixation reagents provided in the Parse Biosciences fixation kit to preserve endogenous RNA profiles and stabilize cellular material. Fixed cells were subsequently washed, counted, and stored under recommended conditions prior to whole-transcriptome split-pool combinatorial barcoding library preparation. Fixed single-cell suspensions were processed using the Parse Biosciences Evercode™ WT Mini scRNA-seq kit. Cells underwent split-pool combinatorial barcoding, in which cells were repeatedly distributed into wells and labeled with unique molecular barcodes. During the first barcoding round, cell-specific barcodes were incorporated and reverse transcription was performed to generate barcoded cDNA from cellular RNA. Subsequent barcoding rounds further labeled cDNA molecules with additional barcode sequences, enabling combinatorial indexing of individual cells. The resulting barcoded cDNA was pooled, amplified, and prepared for next-generation sequencing. Barcoded cDNA was fragmented and processed to generate sequencing-compatible library molecules containing cell-identifying barcode sequences and transcript-derived fragments. Libraries were amplified, purified, and assessed for quality and concentration before sequencing.