sci-RNA-seq3 library construction and sequencing

The paraformaldehyde fixed nuclei were processed similarly to the published sci-RNA-seq3 protocol (11). For paraformaldehyde fixed cells, frozen fixed cells were thawed on 37°C water bath, spun down at 500 × g for 5 min, and incubated with 500 μl PBSI [1 x phosphate-buffered saline (PBS), pH 7.4, 1% bovine serum albumin (BSA), 1% SuperRnaseIn] including 0.2% Triton X-100 for 3 min on ice. Cells were pelleted and resuspended in 500 μl nuclease-free water including 1% SuperRnaseIn. 3 ml 0.1N HCl were added into the cells for 5min incubation on ice (17). 3.5 ml Tris-HCl (pH 8.0) and 35 μl 10% Triton X-100 were added into cells to neutralize HCl. Cells were pelleted and washed with 1 ml PBSR. Cells were pelleted and resuspended in 100 μl PBSI. The following steps were similar with the sci-RNA-seq3 protocol (with paraformaldehyde fixed nuclei) with slight modifications: (i) We distributed 20,000 fixed cells (instead of 80,000 nuclei) per well for reverse transcription (RT). (ii) We replaced all nuclei wash buffer in following steps with PBSI. (iii) All nuclei dilution buffer were replaced with PBS + 1% BSA.