Single cell RNA-seq of mouse megakaryocytes from wild type and Nf-e2 knock out mice

Fetal livers from 8X E13.5 wildtype and 6X Nf2-/- mutant embryos were mechanically dissociated, pooled by genotype and stained for CD41, CD42d, LIN (Ter119,B220). LIN- CD41high CD42d+ Cells were sorted on an Aria Cell Sorter (Becton Dickinson), centrifuged and counted. Fetal livers of 5X E13 wildtype or Nf2-/- embryos were prepared as above and transplanted into UBC-GFP+ recipients subjected to 2X 550cGrays irradiations (0.37 embryonic equivalent/recipient). Mice were screened at 4 weeks for blood platelet content and their GFP status. Bone marrow from the femurs, tibias, hips and humeri of 4 transplanted mice (2X WT donor, 2X Nf2-/- donor) was dissociated, pooled by genotype, filtered and stained for CD41, CD42d, LIN (Ter119, B220). GFP- LIN- CD41high CD42d+ Cells were sorted on an Aria Cell Sorter (Becton Dickinson), centrifuged and counted. 3.9K (Adult) and 6.9K (Fetal Liver) cells were loaded onto a Chromium Controller (10X Genomics). Single cell 3' v2 transcriptome libraries were obtained following manufacturer's instructions. Samples were pooled and sequenced on NextSeq (Illumina) (75bp kit, paired end, where 98bp were sequenced for genomic read).