5 CITE-seq of edited HSCs with cssDNA donor template, Gene expression library
Single-cell mRNA barcoding and library generation were performed following the 10X Genomics protocol from the Chromium Next GEM Single Cell 5 Kit v2 (#1000263) as described in Moiani et al, Nat. Commun. 15, 4965 (2024). Samples from different donors were multiplexed following a previously described cell-hashing protocol. Briefly, 200,000 edited or non-edited HSPCs (D4) were thawed and incubated with Human TruStain FcX Fc Blocking reagent (Biolegend, #422301) for 10 minutes at 4C. Cells were stained with TotalSeq-C Hashtag antibodies diluted at 1/50 in Cell Staining buffer (Biolegend, #420201) for 30 minutes at 4C. After washing, different conditions bearing different hashtags were pooled. 500,000 cells were stained for 30 min at 4C with TotalSeq-C Universal Cocktail V1.0 (Biolegend, #399905) supplemented with 18 additionals TotalSeq-C antibodies at 0.25ug per 500,000 cells. The list of antibody-derived tag (ADT) used for CITE-Seq procedure is documented in supplementary table 2. After washing cells were loaded into Chromium Single-Cell Chip (10X Genomics) at a target capture rate of ~10,000 individual cells per sample. Gene Expression and Cell Surface libraries were checked using a Bioanalyzer High Sensitivity DNA kit (Agilent, #5067-4626) according to the manufacturers recommendations. Libraries generated were sequenced by Institut du Cerveau (ICM) IGenSeq plateformusing a NovaSeq X Plus sequencer from Illumina following 10X recommendations.