GSM8841893: HCC cells with different drugs treatment_Lib2_2nd; Homo sapiens; RNA-Seq
Isolate cell nuclei, perform counting, and suspend them in a reverse transcription mixture (RT mix). For a 96-well plate reaction, 110× RT mix was prepared: 55μL 10 mm dNTP, 484μL RT buffer, 55μL RNA Inhibitor (Vazyme), 55μL Reverse Transcriptase, 341μL WB(with nuclei). The reverse transcription kit was included in the VITAPilote-EFT1200 kit (Cat # R20122124) ordered from M20 Genomics. Both nuclei-RT mix (≤50 000 nuclei, 9 μL per well) and 10 μm well-specific barcoded RT primers (1μL per well) were distributed to each well of the 96-well plate and stirred gently with the pipette tip. The reaction mix was incubated with the thermal cycling: (8 °C for 12 s, 15 °C for 45 s, 20 °C for 45 s, 30 °C for 30 s, 42 °C for 2 min)× 10 cycles, 42 °C for 45 min. After the reaction, all nuclei were collected, mixed, and washed using PBST (PBS, 0.05% Tween 20) three times to remove the residual primers. After washing, nuclei were suspended in TdT mixture (100 000-1 000 000 nuclei per reaction, 39μL nuclei in PBST, 5μL 10× TdT buffer (NEB), 5μL CoCl2 (NEB), 0.5μL 100 mm dATP (Invitrogen), 0.5μL TdT enzyme (NEB)). The TdT reaction mix was incubated at 37 °C for 30min. After reaction, nuclei were washed using PBST three times. The nuclei were counted and diluted to 2000–8000 nuclei μL−1 using OptiPrep (Stem Cell). DNA extension reaction mixture was prepared (for 80μL): 40μL ddH2O, 16μL thermopol buffer, 6μL 10 mm dNTP, 6μL BST 2.0 Warmstart (NEB), 6μL RnaseH (NEB), 6μL USER (NEB). Nuclei, 2 x DNA extension reaction mixture (M20 Genomics, VITAPilote-EFT1200 kit), and barcoded beads were encapsulated into droplets using the microfluidic platform. Circularization was performed to obtain a sequencing nanoball library for MGI DNBSEQ using VAHTS Circularization Kit for MGI (Vazyme, NM201). Library sequencing was performed using DNBSEQ-T7 with paired-end reads of 100 or 150 bp.