title: GSM4762820: Bld WT D0; Mus musculus; RNA-Seq
library_construction_protocol: Bone marrow cells were collected by flushing femurs with ice-cold PBS.  The resulting solution was filtered through a 40 µm nylon mesh and treated with red blood cell (RBC) lysis buffer (BioLegend).   Blood was collected by cardiac puncture.  The cellular fraction was collected into EDTA-containg tubes (Sigma), and erythrocytes were eliminated using RBC lysis.   Heart tissue was collected by incising the right atrium and perfusing 10 mL of ice-cold saline into the left ventricular apex. Heart tissue was then removed and used for one of several assays detailed below. To obtain single cell suspensions for surface immunostaining, flow cytometric analysis, FACS sorting, or single cell RNA-Seq, hearts were enzymatically digested for 1 hour under continuous agitation in 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I, and 60 U/ml hyaluronidase (Sigma) for 1 hour at 37°C, and filtered through a 40 µm nylon mesh cell strainer in FACS buffer (DPBS with 2.5% bovine serum albumin) for enumeration by flow cytometry. For single cell RNA-Seq, the enzymatic digestion was limited to 45 minutes to minimize transcript degradation. Single cell libraries were prepared according to 10X Genomics standard procedures.