GSM4979496: C5_2; Mus musculus; RNA-Seq
Livers were harvested, cut into small pieces and digested in DMEM with 10 mM Hepes, DNase I and Collagenase D at 37°C and 100 rpm for 40 min. Suspensions were passed through 100 mm filters and lymphocytes enriched by 40%/80% percoll gradient (860 x g, 20 min, RT, no breaks). The interphase was washed with PBS and used for sorting. ILC1 and NKs from WT and KO mice were isolated for single cell RNA-sequencing. In order to pool the 4 populations into the same reaction, we labeled with oligo-tagged antibodies targeting ubiquitously expressed cell surface markers (Hashtag antibodies, Biolegend). WT and KO populations were labelled with Hashtags (TotalSeq™-A0301 anti-mouse Hashtag 1 for KO and TotalSeq™-A0302 anti-mouse Hashtag 2 for WT). Cells were furthermore labeled with oligo-tagged antibodies targeting selected surface markers (CD11b, CD127, CD200R, CD27, CD69, CD902, NKp46, TotalSeq™-A, Biolegend) for cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). ILC1 and NKs from WT and KO mice were sorted separately using a FACS Aria III (BD Biosciences) and then pooled all together with the desired proportion. The 4 populations will be identified according to hashtags, antibody tags and gene expression sequences. Immediately after sorting, the cells were encapsulated into droplets using 10X Genomics GemCode Technology and processed following manufacturer's specifications for single-cell RNA-sequencing library preparation. Every cell and every transcript (including oligo tags of Hashtag and CITE-Seq antibodies) were barcoded using a unique molecular identifier (UMI) and cDNA ready for sequencing on Illumina platforms was generated using the Single Cell 3' Reagent Kit v3 (10X Genomics). 3 distinct libraries were generated: a CITE-Seq library, a Hashtag library, and the cDNA library fraction (corresponding to the cell transcriptome). Libraries were quantified by QubitTM 3.0 Fluometer (ThermoFisher), quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent) and sequencing was performed across a NovaSeq 6000 platform (Illumina) in 50bp paired-end mode to reach approximately 150,000 reads per single cell. For adequate read coverage, we sequenced the CITE-Seq library at 10%, the Hashtag library at 5%, and the cDNA library fraction at 85% of a lane according to the manufacturer's recommendations (Biolegend). Chromium single cell 3´v3 (10XGenomics)