Ampure xp beads purification

The cells were dissociated from the plates with Accutase (Sigma, A6964) for 3 minutes at 37°C. ATAC-seq samples and libraries were generated as described previously (Buenrostro et al., 2015 ) except the nuclei were prepared using 100 μl of ice cold Nuclei EZ lysis buffer (Sigma, N3408). The nuclei pellets were resuspended in 10 μl H₂O and nuclei were counted. The tagmentation reaction was performed with 50 thousand nuclei and 2.5 μl of Tn5 transposase (0.5 μM) in 25 μl reaction volumes for 30 mins at 750 rpm at 37°C. The tagmented genomic DNA was purified by using miniElute Reaction Cleanup kits (QIAGEN, 28204) and eluted in 10.5 μl. The libraries were generated by 9 cycles of PCR reaction using adaptor primers (Nextera Index kit; Illumina, FC-121-1012) and NEBNext high fidelity 2x PCR master mix (NEB, M0541), followed by two-sided size selection by Ampure XP beads purification (0.4x reaction volume then 1.2x reaction volume; Beckman Coulter Agencourt, A63881). The typical yield is between 150-300 ng. The sequencing was performed on an Illumina Next-seq genome analyzer according to the manufacturer’s protocols.

cDNA (40 fmol) amplified from five independent reactions of each cell line or tumor sample were combined in equal proportions for Nanopore sequencing. The combined cDNA was prepared by Q20+ ligation sequencing kit (Oxford Nanopore). In brief, 48 µL cDNA was incubated with 3.5 µL NEBNext formalin-fixed paraffin-embedded (FFPE) DNA Repair Buffer, 2 µL NEBNext FFPE DNA Repair Mix, 3.5 µL Ultra II End-prep reaction buffer, and 3 µL Ultra II End-prep enzyme mix (NEB) at 20°C for 5 minutes and 65°C for 5 minutes followed by AMPure XP beads purification (Beckman Coulter). cDNA (60 µL) eluted from AMPure XP bead purification was ligated with sequencing adapters by incubation with 5 µL Adapter Mix H, 10 µL NEBNext Quick T4 DNA Ligase (NEB), and 25 µL Ligation Buffer (Oxford Nanopore) at room temperature for 10 minutes. The cDNA was then purified one additional time by AMPure XP beads with L fragment buffer.

Fifty nanoliter of SMART RT-PCR reagents (Takara Bio) were dispensed to all selected wells and full-length cDNA was amplified in-chip for 24 cycles using SMARTScribe reverse transcriptase according to manufacturer's instructions (Figure 1B). Barcoded amplicons were collected into a tube by centrifugation and off-chip purified with the NucleoSpin Gel and PCR Clean-up kit (Machery-Nagel, Düren, Germany) followed by an AMPure XP beads purification (Beckman Coulter, Brea, USA). cDNA synthesis, purification and size selection were checked on a Bioanalyzer using the High sensitivity DNA kit [Agilent Technologies, Santa Clara (CA), USA]. The resulting cDNA library was split for either TRA/TRB amplification or whole transcriptome amplification.