Single-cell multi-omics profiling

Single cells were individually transferred into 200-μl tubes using a mouth pipette. The single cells were lysed in 7 μl of soft buffer (500 mM KCl, 100 mM Tris-HCl (pH = 8.3), 1.35 mM MgCl₂, 4.5 mM DTT, 0.45% Nonidet P-40 (Roche, 11332473001), 0.18 U SUPERase-In (Applied Biosystems, AM2694), and 0.36 U RNase-inhibitor (Applied Biosystems, AM2682) for 30 min at 4 °C, and then the lysate was vortexed for 1 min at room temperature. All RNAs were released, whereas the nucleus remained intact. The lysed single cell was then centrifuged at 1 000× g for 5 min to leave the nucleus at the bottom of the tube. The 4 μl of lysis product supernatant was carefully removed and added to another 200-μl tube containing spike-in RNA (ERCC, Ambion) and reverse transcriptase. This fraction was used for transcriptome analyses, whereas the remaining 3 μl of lysis solution (containing the nucleus) was used for genome (CNVs) and methylome analyses. The upper 4 μl of lysis solution was reverse-transcribed with poly T primers, and the cDNA was amplified as previously described^{29 (link)}. Protease was added to the bottom 3 μl lysis solution, and the DNA was added with 60 fg of unmethylated lambda DNA (Fermentas). The released naked DNA was then digested and bisulfite-converted using the scRRBS method^{19 (link)}.