{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Xi Chen"],"instrument_platform":["NextSeq 550"],"study_type":["scATAC-seq"],"organism":["Mus musculus"],"species":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15131"],"description":["Multi-modal profiling of different molecular layers from the same single cell enables more comprehensive characterisations of cellular heterogeneity compared to conventional single-modality approaches. A key example is co-detection of chromatin accessibility and gene expression that offers the opportunity to investigate the cell type-resolved gene regulatory mechanisms. Here, we described a sensitive and robust protocol of in situ SHERRY after ATAC-seq (ISSAAC-seq) for the concurrent measurement of chromatin accessibility and gene expression from the same single nucleus. The method begins with dual Tn5 tagging of open chromatin regions and DNA/RNA hybrid after reverse transcription that take place in bulk nuclei. Then various single-nucleus isolation strategies can be used based on the experimental purpose of the user. The protocol is highly modular with flexible throughputs ranging from several hundreds to tens of thousands of nuclei. The generated data are of high quality in both modalities. The entire workflow can be finished within 1 or 2 days, and the procedures works on multiple different platforms."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - PBMCs from a healthy donor were isolated using Lymphoprep (STEMCELL, catalog no. 07801). Following isolation, PBMCs were subjected to red blood cell lysis, washing and counting. PBMC aliquots were cryopreserved in 90% FBS + 10% DMSO and stored in liquid nitrogen. After thawing, dead cells were removed using Dead Cell Removal Kit (Miltenyi Biotec, catalog no. 130-090-101). Then 1 × 106 PBMCs were crosslinked in 1 ml of freshly prepared glyoxal fixation buffer (3% glyoxal, 0.75% glacial acetic acid, pH 5.0) at room temperature for 7 min, washed twice with 1 ml of DPBS-0.5% w/v BSA and held on ice.","Nucleic Acid Extraction - Nuclei acid was extracted using 0.8× VAHTS DNA cleaning beads and elute in 20 μl Nuclease-free Water.","Sample Collection - An adult mouse brain cortex was dissected, snap-frozen in liquid nitrogen and stored at -80 °C. For single nucleus isolation,  frozen cortex (2-3 mm3) was placed into a prechilled 1 ml of Dounce homogenizer with 1 ml of homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 8.0, 1 μM DTT, 1× Protease Inhibitor Cocktail (Roche,  catalog no. 11697498001), 0.4 U/μl RNasin (Promega, catalog no. N2111) and 0.1% (v/v) Triton X-100). Tissue was homogenized with five strokes of the loose pestle, followed by ten strokes of the tight pestle. The sample was centrifuged at 100 g, 4 °C for 1 min to remove large debris. The supernatant was filtered through a prechilled 2-ml round bottom tube with cell-strainer cap (Falcon), and the effluent was transferred to a prechilled 1.5-ml tube, centrifuged at 1,000 g, 4 °C for 3 min. The pellet was resuspended in 1 ml of ice-cold NIM2 buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 8.0, 1 μM DTT, 1× Roche Protease  Inhibitor Cocktail and 0.4 U/μl RNasin) and centrifuged at 1,000 g, 4 °C for 3 min. Nuclei were then resuspended in 1 ml of ice-cold PBSI (1% (w/v) BSA, 1 μM DTT  and 0.4 U/μl RNasin).","Sequencing - Library was sequenced on Illumina X-Ten or NovaSeq 6000 according to the manufacturer's instructions","Library Construction - 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."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Xi Chen"],"additional_accession":[]},"is_claimable":false,"name":"Single-nucleus chromatin accessibility and gene expression co-profiling by ISSAAC-seq","description":"Multi-modal profiling of different molecular layers from the same single cell enables more comprehensive characterisations of cellular heterogeneity compared to conventional single-modality approaches. A key example is co-detection of chromatin accessibility and gene expression that offers the opportunity to investigate the cell type-resolved gene regulatory mechanisms. Here, we described a sensitive and robust protocol of in situ SHERRY after ATAC-seq (ISSAAC-seq) for the concurrent measurement of chromatin accessibility and gene expression from the same single nucleus. The method begins with dual Tn5 tagging of open chromatin regions and DNA/RNA hybrid after reverse transcription that take place in bulk nuclei. Then various single-nucleus isolation strategies can be used based on the experimental purpose of the user. The protocol is highly modular with flexible throughputs ranging from several hundreds to tens of thousands of nuclei. The generated data are of high quality in both modalities. The entire workflow can be finished within 1 or 2 days, and the procedures works on multiple different platforms.","dates":{"release":"2025-06-01T00:00:00Z","modification":"2025-11-20T20:53:11.287Z","creation":"2025-05-20T11:04:36.123Z"},"accession":"E-MTAB-15131","cross_references":{"ENA":["ERP172751"],"Biostudies":["E-MTAB-11264"],"EFO":["EFO_0002944","EFO_0004170","EFO_0010891","EFO_0005518","EFO_0004184"]}}