HH-seq workflow

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The fixed nuclei were counted and suspended in reverse transcription mixture (RT mix). For a 96-well plate reaction, 110 x RT mix were prepared: 55 µL 10 mM dNTP, 484 µL RT buffer, 55 µL RNA Inhibitor (Vazyme), 55 µL Reverse Transcriptase, 341µL WB (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/well) and 10 µM well-specific barcoded RT primers (1 μL/well) were distributed to each well of the 96-well plate and stirred gently with the pipette tip. The reaction mix were 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) x 10 cycles, 42°C for 45 min. After reaction, all nuclei were collected, mixed and washed using PBST (PBS, 0.05% Tween 20) for 3 times to remove the residual primers.

After washing, nuclei were suspended in TdT mixture (1,000,000 nuclei per reaction, 39 µL nuclei in PBST, 5 µL 10 x TdT buffer (NEB), 5 µL CoCl2 (NEB), 0.5 µL 100mM dATP (Invitrogen), 0.5 µL TdT enzyme (NEB)). The TdT reaction mix was incubated at 37 ºC for 30 min. After reaction, nuclei were washed using PBST for 3 times. The nuclei were counted and diluted to 2,000 nuclei/µL using OptiPrep™ (Stem Cell).

Nuclei, 2 x DNA extension reaction mixture (M20 Genomics) and barcoded beads were encapsulated into droplets using the microfluidic platform. The droplets (50 µL per tube) were incubated at 37°C for 1 hour, 50°C for 30 min, 60°C for 30 min, 75°C for 20 min. Then the droplets were broken by mixing with equal amounts of 20% PFO (1H,1H,2H,2H-Perfluoro-1-octanol, Sigma). The supernatant was collected after centrifugating and purified with 1.2 x DNA Clean Beads (Vazyme) and eluted in 40 μL ddH2O. Two rounds of PCR were performed to amplify cDNA and add sequence adapters. The amplified libraries were purified with 0.8 x DNA Clean Beads and quantified using Qubit (Invitrogen). 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.

All processed scRNA-seq data was available at figshare https://figshare.com/articles/dataset/pan-cancer_using_snHH-seq/22256212.