Illumina NovaSeq 6000 paired end sequencing; Single Cell Multi-Omics Profiling Using ISSAAC-seq
Single Cell Multi-Omics Profiling Using ISSAAC-seq
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. hESC H1 cells were cultured according to standard procedures in mTeSR1 (STEMCELL) on Corning Matrigel hESC-Qualified Matrix (Corning). hESCs at 80% confluence were harvested using StemPro Accutase cell dissociation reagent (Thermo Fisher).   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. FACS library: In this case both RNA and ATAC have four files: R1 (variable length), R2 (variable length), I1 (8 bp) and I2 (8 bp). The combination of I1 and I2 are the cell barcodes (8 + 8 = 16 bp).For RNA, the first 10 bp of R1 are UMIs, and the rest are discarded. R2 is the cDNA read. For ATAC, both R1 and R2 are gDNA reads. Droplet strategy: both RNA and ATAC have three files: R1 (151 bp), R2 (151 bp) and I2 (16 bp). The cell barcodes are basically I2 (16 bp). For RNA, the first 10 bp of R2 are UMIs, and the rest are discarded. R1 is the cDNA read. For ATAC, both R1 and R2 are gDNA reads. For Chromatin tagmentation (ATAC-seq), nuclei were resuspended in 50 μl of Tn5-S5/S7 tagmentation mix containing 33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, 16% dimethylformamide (DMF, sigma), 0.01% digitonin, 1.2 U/μl RiboLock Rnase inhibitor, 0.4 U/μl SUPERaseIn (Thermo Fisher), 0.8 U/μl RnaseOUT (Thermo Fisher), and 5 μl Tn5-S5/S7 transposome complex. The tagmentation reaction was done on a thermomixer at 800 rpm, 30 °C, 30 min. The reaction was then stopped by adding equal volume (50 μl) of tagmentation stop buffer (10 mM Tris-HCl pH 7.8, 20 mM EDTA, pH 8.0, 2% BSA). Then proceed to the SHERRY section. The SHERRY (Sequencing HEteRo RNA-DNA-hYbrid, doi: 10.1073/pnas.1919800117) method was used for the RNA portion of the library. Briefly, Aater chromatin tagmentation, nuclei were spun down at 1000g, 4°C, 3 min, and washed twice with 200 μl of ice-cold 0.5× DPBS-0.5% BSA containing 0.2 U/μl RiboLock Rnase inhibitor. Nuclei were resuspended in 100 μl of reverse transcription mix containing 0.5 mM dNTP, 10 U/μl Maxima H minus reverse transcriptase, 0.8 U/μl RiboLock Rnase inhibitor, 0.2 U/μl SUPERaseIn, 0.4 U/μl RnaseOUT, 12% PEG8000, 50 mM Tris-HCl, pH 8.0, 75 mM sodium chloride, 3 mM magnesium chloride, 10 mM dithiothreitol, and 2 μM TruseqR1_oligo_dT for the plate-based workflow or 2 μM TruseqR2_oligo_dT for the 10x droplet workflow. The in-situ reverse transcription reaction was performed as follows: 50°C 10 min; then 3 cycles of: 8°C 12s, 15°C 45s, 20°C 45s, 30°C 30s, 42°C 2 min, 50°C 3 min; followed by a final step at 50°C for 5 min. After reverse transcription, nuclei were spun down at 800g, 4°C, 5 min, and washed twice with 200 μl of ice-cold 0.5× DPBS-0.5% BSA, then resuspended in 50 μl of tagmentation mix containing 33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, 16% dimethylformamide (DMF, sigma), 0.01% digitonin, and 1.5 μl of Tn5-S7/S7 transposome for the plate-based workflow or 1.5 μl of Tn5-S5/S5 for the 10x droplet workflow. The tagmentation reaction was done on a thermomixer at 800 rpm, 37 °C, 30 min. The reaction was then stopped by adding equal volume (50 μl) of tagmentation stop buffer (10 mM Tris-HCl pH 7.8, 20 mM EDTA, pH 8.0, 2% BSA). After RNA/DNA hybrid tagmentation, nuclei were spun down at 800g, 4°C, 5 min, and washed twice with 200 μl of ice-cold 0.5× DPBS-0.5%BSA. Then resuspended in 50 μl of reaction mix containing 0.5 mM dNTP, 8 U/μl Maxima H minus reverse transcriptase, 2 U/μl Thermolabile Exonuclease I (NEB), 50 mM Tris-HCl, pH 8.0, 75 mM sodium chloride, 3 mM magnesium chloride, and 10 mM dithiothreitol. EXO I digestion and gap fill-in reaction was done on a thermomixer at 800 rpm, 37 °C, 15 min.