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Fresh frozen tissues were homogenized in 1 mL of homogenization buffer using a freezing multi-sample tissue grinding system (TissueLyser-24, Jingxin Industrial Development, China). Homogenization buffer contained 250 mM sucrose, 25 mM KCl, 25 mM MgCl2, 10 mM Tris buffer, 1 mM DTT, 1 X protease inhibitor, 0.4 U/mL RNaseIn (Thermo Fisher Scientific), 0.4 U/mL Superasin (Thermo Fisher Scientific), 0.1% Triton X-100 (v/v), 1 mM PI (Thermo Fisher Scientific), and 10 ng/mL Hoechst 33342 (Thermo Fisher Scientific). After being passed through a 40-μm filter (BD Falcon), nuclei were centrifuged at 500 × g for 5 min and resuspended in PBS supplemented with 0.3% BSA (Gemini), 0.4 U/mL RNaseIn (Promega N2615) and 0.4 U/mL Superasin (Invitrogen, AM2694). Nuclei positive for Hoechst 33342 and PI were sorted by FACS (BD Influx) and counted using a dual fluorescence cell counter (Luna-FLTM, Logos Biosystems). |
RNA-Seq |
TRANSCRIPTOMIC SINGLE CELL |
PolyA |
PAIRED
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