GSM7784884: AngII_CD34 cells; Mus musculus; RNA-Seq
After collecting the heart of ApoE-/- or AngII-operated mice hearts, the tissue was minced into fine pieces using a scalpel and transferred into a glass vial with 1.5 ml of cold digestion buffer (Liberase, Roche). The tissues were digested by either using a shaking (100 rpm) 37°C waterbath for 15 min. The obtained cell suspension was gently pipetted up and down (10 x) and transferred onto a 70μm cell strainer placed on top of a 50ml Falcon tube. The tissue was gently rubbed through the strainer using the plunger of a 1ml syringe, after which the strainer was rinsed with 8.5ml of DMEM to obtain a total volume of 10 ml, which was centrifuged for 6 min at 4°C at 300 xg. After the supernatant was removed, non-cardiomyocytes samples pelleted cells were suspended in red blood cell lysis buffer (Miltenyi Biotec) to lyse red blood cells. After washing with PBS, cells were filtered with 40 μm filter and then centrifuged at 300xg for 5 minutes. Cells resuspended in PBS were stained with LIVE/DEAD Fixable Near-IR (APC/Cy7 channel) Dead Cell Stain Kit (Invitrogen; L34975, 1:1000) and Hoechst 33342 (Invitrogen; H3570, 1:1000) for 20 minutes. Unstained cells and cells staining with only 1 fluorochrome prepared concomitantly served as control. After 1 wash in PBS for 5 minutes, cells were resuspended in PBS and then sorted by Flow Cytometer. Nucleated live cells (Hoechst+/APC/Cy7− population) were sorted into PBS with 0.04% BSA for subsequent scRNA-seq. For isolation of single CD34+ cells from the heart, a similar protocol was used as describe above. RNA libraries were prepared with Chromium™ Single Cell 3′ Reagent Kit v3 chemistry (10× Genomics) according to the manufacturer's protocol.