GSM8960400: IL7RA+, scRNA; Mus musculus; RNA-Seq
Up to 100.000 ex vivo sorted BM progenitors (LSK, FLT3+, IL7Ra+ and CD19+) were spun down (300xg, 5 min 4 °C) and washed in PBS/0.4%BSA. . Nuclei were isolated according to Nuclei Isolation for Single Cell Multiome ATAC + GEX. Sequencing (CG000169, Rev C, 10x Genomics). Briefly, cells were resuspended in 100 µl chilled Lysis buffer (10 mM Tris-Hcl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, 0.01 % Digitonin (Invitrogen, BN2006), 1% BSA, 1 mM DTT, 1 U/µl RNase inhibitor (Roche, 3335402001) and incubated 3 min on ice. Nuclei were washed 3x in 1 ml chilled Wash buffer (10 mM Tris-Hcl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.1 % Tween-20, 1 mM DTT, 1 U/µl RNase inhibitor) before resuspension in 10-20 µl chilled diluted Nuclei buffer (1x Nuclei buffer (10x Genomics, 2000207), 1 mM DTT, 1U/µl RNase inhibitor). Nuclei concentrations were determined using TC20 automated cell counter (BioRad). Single cell ATAC and Gene Expression libraries were constructed according to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338, Rev F, 10x Genomics) using reagent from the Chromium Next GEM Single Cell Multiome ATAC+ Gene Expression Reagent Bundle (10x Genomics, 1000285). Briefly, nuclei were transposed in bulk solution followed by subsequent GEM generation and barcoding in a Chromium Next GEM Chip J Single Cell (10x Genomics, 1000230) using a Chromium controller (10x Genomics). Post-GEM cleanup followed by Pre-Amplification of barcoded transposed DNA and cDNA were performed prior to ATAC and Gene Expression Library generation with Singel Index Kit N (10x Genomics, 1000212) and Dual Index Kit TT (10x Genomics, 1000215) respectively. Library quality control was performed with Qubit® dsDNA HS Assay Kit & assay tubes ThermoFisher Scientific, Q32854/Q32856) and Agilent 2100 Bioanalyser with the High Sensitivity DNA Kit (Agilent, 5067-4626). Libraries were subjected to paried-end dual-index sequencing (ATAC: Read 1N 50 cycles, i7 Index 8 cycles, i5 24 cycles, Read 2N 49 cycles, RNA: Read 1 28 cycles, i7 Index 10 cycles, i5 Index 10 cycles, Read 2 90 cycles) on a Nextseq 2000 (Illumina) using NextSeq2000 P3 100 cycles med PhiX (Illumina, 20040559) according to manufacturer's instructions.