GSM7871879: SS2nTregAct_3; Mus musculus; RNA-Seq
Live cells were sorted using PI staining, then 5 × 104 cells were transferred to a low-binding centrifuge tube and resuspended in 50 μL of pre-chilled Lysis Buffer. After 20 times pipetting, cells were incubated on ice for 10 minutes for lysis. RNA-seq The RNA-seq library preparation was performed by a modified SMART-seq2 protocol49,51. Briefly, RNA was added into 4 µl elution mix made of 1 µl RT primer (10 µM), 1 µl dNTP mix (10 mM each), 1 µl RNase inhibitor (4 U/µl), and 1 µl H2O. Eluted samples were incubated at 72 °C for 3 min and immediately placed on ice. Each sample was added with 7 µl reverse transcription (RT) mix made of 0.75 µl H2O, 0.1 µl Maxima RNase-minus RT (Thermo Fisher Scientific, #EP0741), 2 µl 5x Maxima RT buffer, 2 µl Betaine (5 M, Sigma-Aldrich, B0300), 0.9 µl MgCl2 (100 mM), 1 µl TSO primer (10 µM), 0.25 µl RNase inhibitor (40 U/µl).The RT reaction was incubated at 42 °C for 90 min and followed by 10 cycles of (50 °