GSM9427936: EOF+EP+DMF-14; Mus musculus; RNA-Seq
The capillary tip was filled with 1.5 μL of mixed solution (2.5 μM biotinylated-oligo-dT primer, 2.5 mM dNTP (AD101–12, TransGen Biotech, Beijing, China), 0.4 U·μL−1 Murine RNase Inhibitor (GMP4102PA-01, Vazyme Biotech Co., Ltd., Nanjing, China)), and then connected to the work electrode of the ultra-micro extraction instrument (Rayme Biotechnology Co., Ltd., Jiangsu, China). The counter electrode and the reference electrode were immersed in the culture medium within the confocal dish. The cells to be extracted were visualized by microscope and the preloaded capillary was gradually moved downward until it touched the cytoplasm of the selected cell. The tip of the capillary was then inserted into the cytoplasm, with a deformation observed in the selected cell. By setting the applied voltages and the duration of current application, a precisely controlled volume was extracted from the cell. After that, the capillary was retracted out of the cell, taken out of the extraction instrument and placed at -80°C for RNA storage. Once it was required to undergo the RNA library construction, the capillary was connected to the work electrode again. The cellular contents within the capillary were injected into the top plate of the DMF device, enabling the subsequent RNA library processing on the DMF system. The lysis buffer (2.5 μM biotinylated-oligo-dT primer, 2.5 mM dNTP, 0.4 U·μL−1 Murine RNase Inhibitor and 0.2% Triton-X-100(A110694, Sangon Biotech, Shanghai, China) was introduced and mixed with the cellular contents at 72°C for 3 minutes for complete RNA release. Then the reverse transcription (RT) mix (1.7 μM biotinylated-TSO oligo, 5 × HiScript II RT buffer (Vazyme Biotech Co., Ltd., Nanjing, China), 1.7 U·μL−1 Murine RNase Inhibitor, 19 U·μL−1 HiScript II Reverse Transcriptase (R201, Vazyme Biotech Co., Ltd., Nanjing, China) and 0.05% Pluronic F68 (24040032, ThermoFisher, America)) was introduced to initiate the RT reaction at 42°C for 90 minutes, which was stopped by heat at 70°C for 15 min. After that, the PCR mix (2 × KAPA HiFi HotStart ReadyMix (KK2602, KAPA, America), 0.06 μM Biotinylated-ISPCR oligo and 0.05% Pluronic F68) was introduced to amplify the cDNAs (95°C 3 minutes, 25 cycles of 98°C 30 s, 65°C 45 s and 72°C 6 minutes, followed by a final step at 72˚C for 5 min). The products were recovered from the DMF device into a 200-μL eppendorf tube, purified with 0.6 × DNA clean beads (Vazyme Biotech Co., Ltd., Nanjing, China) and quantified by qubit 3.0. Then 1 ng of the products were utilized and processed by sequencing-ready library construction kits (TD503, Vazyme). The library was purified twice with 0.6 × DNA clean beads (N411, Vazyme Biotech Co., Ltd., Nanjing, China) and sequenced by Novaseq500 according to the manufacturer's instructions.