Agilent tapestation high sensitivity d5000 screentape system

ScRNA-Seq was performed using the 10X Genomics platform (10X Genomics, Pleasanton, CA). FACS sorted cells from either mice or organoid cultures were encapsulated with a 10X Genomics Chromium Controller Instrument using the Chromium™ Single Cell A Chip Kit. Encapsulation, reverse transcription, cDNA amplification, and library preparation reagents are from the Chromium™ Single Cell 3’ Library & Gel Bead Kit v2. Briefly, single cells were resuspended in PF10 at a concentration of 1000 cells μ⁻¹. The protocol was performed as per 10X Genomics protocols without modification (chromium single cell 3 reagent kits user guide v2 chemistry). Total cDNA and cDNA quality following amplification and clean-up was determined using a QubitTM dsDNA HS assay kit and the Agilent TapeStation High Sensitivity D5000 ScreenTape System. Library quality pre-sequencing was determined using Agilent TapeStation and QPCR prior to sequencing. TapeStation analysis and library QPCR was performed by the Biopolymers Facility at Harvard Medical School. Libraries were sequenced using an Illumina NextSeq500 using paired-end sequencing with single indexing (Read 1 = 26 cycles, Index (i7) = 8 cycles, and Read 2 = 98 cycles). Reads were aligned to the mm10 reference genome and count matrices were generated using CellRanger3.0.0 (10X Genomics).