title: Single-nucleus chromatin accessibility and gene expression co-profiling by ISSAAC-seq
library_construction_protocol: Cells were cultured according to standard procedures in RPMI 1640 for K562 and GM12878 cells or DMEM for HEK293T and NIH3T3 cells, supplemented with 10% FBS (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. HEK293T and NIH3T3 cells were digested with 0.25% Trypsin for preparing single-cell suspension. Nuclei acid was extracted using 0.8× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water. For final ATAC+RNA library amplification in the plate-based workflow, 15 μl of purified pre-library was mixed with 2.5 μl RNA_plate_S5xx primer, 2.5 μl ATAC_plate_S5xx primer, 5 μl illumina P7 primer, and 25 μl Q5 High-Fidelity 2× Master Mix. PCR was performed as follows: 98 °C 1 min; 10 cycles of: 98 °C 20 s, 63 °C 20 s, 72 °C 1 min; 1 cycle of 72 °C 5 min; then hold at 10 °C. Final library was purified using 1.2× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water. For final ATAC library amplification in the 10x droplet workflow, 40 μl of purified 10x ATAC pre-library was mixed with 50 μl Q5 High-Fidelity 2× Master Mix, 5 µl Illumina P5 primer (10 µM), and 5 µl ATAC_droplet_N7xx primer (10 µM). PCR was performed as follows: 98 °C 1 min; 7 cycles of: 98 °C 20 s, 63 °C 20 s, 72 °C 20 s; 1 cycle of 72 °C 1 min; then hold at 10 °C. Final ATAC library was purified using 1.0× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water. For final RNA library amplification in the 10x droplet workflow, 40 μl of purified 10x RNA pre-libraries was mixed with 50 μl Q5 High-Fidelity 2X Master Mix, 5 µl Illumina P5 primer (10 µM), and 5 µl RNA_droplet_N7xx primer (10 µM). PCR was performed as follows: 98 °C 1 min; 7 cycles of: 98 °C 20 s, 63 °C 20 s, 72 °C 20 s; 1 cycle of 72 °C 1 min; then hold at 10 °C. Final RNA library was purified using 0.8× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water. These basically a combination of ATAC-seq and SHERRY protocols.