GSM7575143: TAC-Osi-1, snRNA-Seq; Mus musculus; OTHER
All equipment were washed by RNase Zap before use. Remove heart tissue from -80°C storage and place in pre-chilled petri dish filled with cold Dissociation Buffer (5mM CaCl2, 3mM MgAc, 2mM EDTA, 0.5mM EGTA, 10mM Tris-HCl, 1mM DTT, 0.4% Triton x-100 in RNase-free water) on ice, cut it into small pieces, then transfer to Dounce Homogenizer contains 1mL dissociation buffer, homogenize for 5-10 times, with loose and tight heads, respectively. After homogenization, incubate the lysate on ice for 5 minutes, then centrifuge at 40g for 2minutes at 4°C to remove remaining large fragments. Filter lysate through 70µM and 40µM Flowmi filters (Sigma), centrifuge at 1000g for 5 minutes at 4°C to collect the nuclei. Resuspend the pellet in 1.5mL Sucrose Buffer (1M sucrose, 3mM MgAc, 10mM Tris-HCl, 1mM DTT in RNase-free water), then centrifuge another 5 minutes at 1000g. Resuspend nuclei with 100µL Resuspension Buffer (2%BSA, 1mM DTT in 1xPBS), aliquot 10µL of it for imaging in Trypan Blue. After calculation of nuclei number, the resuspension is centrifuged at 1000g for 5 minutes at 4°C, discard the supernatant and store the nuclei in -80°C until further use. Eight mouse snRNA-Seq libraries were prepared from four treatment groups (n=2 per group) – Sham, Osimertinib (Osi), Transverse aortic constriction (TAC), and TAC-Osi – using Next GEM 10x Genomics Chromium Single Cell 3' Reagent Kit v3.1 chemistry, and sequenced on Illumina NovaSeq 6000 at Translational Genomics Research Institute (TGen), an affiliate of City of Hope.