GSM7502462: WT-R3; Mus musculus; RNA-Seq
Hearts were harvested on day 7 post injury and used for single nuclei preparation for 10X genomics sequencing. Briefly, the heart tissue was minced with razor blade and gently homogenized by Dounce homogenizer containing 1.5 ml of chilled lysis buffer (in mM) (10 Tris-HCl (pH 7.4), 10 NaCl, 3 MgCl2, 0.1% NP-40). Then the homogenate was filtered through a 40 µm cell strainer and centrifuged at 500 g for 5 min at 4 °C. The pellet was resuspended in 1 ml nuclei wash buffer (1x PBS with 2% BSA and 0.2 U/mL RNase inhibitor). The suspension was filtered through a 20 µm cell strainer and centrifuged at 500 g for 5 min at 4 °C. The nuclei pellet was resuspended in 300 mL nuclei wash buffer followed by library preparation. Equal number of cells from 3 mouse hearts were mixed for each group. Single nuclei gene expression libraries were created using Chromium Next GEM Single Cell 3' (v3.1 Chemistry) (10x Genomics), Chromium Next GEM Chip G Single Cell Kit (10x Genomics), and Single Index Kit T Set A (10x Genomics) according to the manufacturer's instructions. Briefly, samples were loaded to target10,000nuclei to form GEMs and barcode individual cells. GEMs were then cleaned cDNA and libraries were also created according to manufacturer's instructions. Library quality was assessed using 4200 TapeStation System and D1000 ScreenTape (both from Agilent) and Qubit 2.0 (Invitrogen) for concentration and size distribution.