title: GSM6206566: PREP0031_CHaas10169A_B06v1_P_DsR_20200312_S3; Mus musculus; RNA-Seq
library_construction_protocol: To remove contaminating RBCs prior to sample isolation, all mice were first perfused with 10ml of 5mM EDTA/PBS at a flow rate of 5ml/min and then with 10ml PBS (without Ca/Mg) at a flow rate of 5ml/min. Samples were isolated from distinct proliferative microenvironments in the calvarium by a combination of plasma mediated laser ablation and micropipette aspiration (Image-seq samples, see manuscript for details). Dissociation of tissues into single cells: All Image-seq and control samples were collected in Media 199 supplemented with 2% (v/v) FBS. Samples were pipetted up and down to generate a single cell suspension and 1ml of PBS (no Ca/Mg) was added to the cell suspension. Samples were incubated for 10min with 0.1µg of DAPI, and AML cells were sorted into individual wells of a 96well PCR plate filled with lysis buffer based on their GFP fluorescence. Samples were sealed and spun down immediately after sorting, flash frozen and stored at -80deg Celsius. Libraries were prepared using the MANTIS Liquid Handler (Formulatrix) and the Biomek FXP Single Arm System with Span-8 Pipettor (Beckman Coulter, A31843). Full-length cDNA was prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio USA, Inc.) and sequencing libraries prepared using the Nextera XT DNA library preparation kit (Illumina).