GSM7910955: wt_Ccnt1_MI_r1; Mus musculus; RNA-Seq
Whole heart was dissected into 1mm pieces on ice with scalpel in 8ml of buffer (300 mM sucrose, 5mM CaCl2, 5 mM Mg acetate, 2mM EDTA, 0,5mM EGTA, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1× protease inhibitor, 0.4 U μl−1 RNaseIn in nuclease-free water) and transferred into the dounce, homogenised over 10 strokes with pestle A and 8 with pestle B on ice, strained through 40um cell strainer and centrifuged 500g 5min at 4C. Further supernatant was discarded, and pallet was resuspended in 0,5ml 1M sucrose buffer (1 M sucrose, 5 mM Mg acetate, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1× protease inhibitor, 0.4 U μl−1 RNaseIn in nuclease-free water) and layered over 1ml 1M sucrose buffer, centrifuged at 1000g 5min at 4C and resuspended in 0,5ml PBS+ 2%BSA + SUPERaseIn with 1:1000 Hoechst. FACS sorting of nuclei was performed using the SH800Z cell sorter (Sony), after which nuclei were collected by centrifuging 600g 8min 4C, counted using hemocytometer and dilute to yield 10 000 nuclei in 43ul 1x PBS, 2%BSA, 0.2 U μl−1 SUPERaseIn. Nuclei were submitted to the Cancer Research UK (CRUK) Cambridge Institute for 10x Chromium library preparation using V3 chemistry and sequencing on a NovaSeq 6000 (Illumina).