GSM8658069: Splenic and bone marrow NK cells from CML mouse 3 - Cell Hashing; Mus musculus; RNA-Seq
For pooling purposes, bone marrow and spleen samples from (2) control and (3) CML mice were stained with TotalSeq-B hashtaging antibodies 1 (bone marrow of each mouse) and 2 (spleen of each mouse) according to the protocol provided by BioLegend. Live singlets gated on host CD45.1+ NK cells (CD3-IL2Rb+NK1.1+CD49b+) were sorted in DPBS 0.04% BSA before loading onto 10x Genomics instrument according to the manufacturer's protocol. Tissues from each mouse were pooled together, while each individual mouse represented one sequencing sample. The recovery of bone marrow-derived NK cells of CML1 mouse was insufficient to proceed with scRNA-seq. Gene expression and feature barcode libraries for single-cell RNA-seq were prepared using the Chromium Next GEM Single Cell 3' Gene Expression v3 with modifications for hashtag libraries. Quality control for amplified cDNA libraries and sequencing libraries was performed using a BioAnalyzer High Sensitivity DNA kit (Agilent). GEX and Cell Hashing libraries were sequenced on Illumina NextSeq 500 with a sequencing depth of at least 50,000 reads per cell for 3' GEX and 5,000 reads per cell for feature barcode.