title: GSM8189709: ME13_20um_RNA; Mus musculus; RNA-Seq
library_construction_protocol: The workflow of simultaneous profiling of chromatin accessibility or a panel of cell surface proteins with two histone modifications and gene expression on the same tissue cryosections is shown schematically in Fig. 1a and Extended Data Fig. 1a-b. For joint analysis of chromatin accessibility, two histone modifications, and transcriptome (Extended Data Fig. 1a, Methods), a frozen tissue section was first fixed with formaldehyde and in situ Tn5 transposition was performed to insert a unique barcoded ligation linker to the accessible DNA loci. The same tissue section was then incubated with two primary antibodies targeting different histone modifications simultaneously, such as the combination of H3K27me3 with H3K27ac or H3K4me3. Afterwards, species specific nanobody-Tn5 fusion proteins loaded with unique barcoded ligation linkers were added to enable the demultiplexing of different histone modification loci. Next, the tissue section was incubated with a biotinylated DNA adaptor containing a universal ligation linker and poly-T sequence that binds to the messenger RNA (mRNA) to perform in situ reverse transcription (RT). Next, barcodes A (A1-A50) and barcodes B (B1-B50) were sequentially flowed over the tissue using microchannels and were conjugated to the universal ligation liker via templated ligation, which formed a two-dimensional grid of spatially barcoded tissue pixels (n =2,500), allowing all of modalities from the same pixel share the same spatial barcodes. Finally, barcoded complementary DNA (cDNA) and genomic DNA (gDNA) fragments were released by reverse crosslinking. cDNAs were separated from gDNA by streptavidin beads. Sequencing libraries for cDNAs and gDNA were separately constructed. For co-profiling of protein, gene expression, and histone modifications (Extended Data Fig. 1b, Methods), the fixed frozen tissue section was first incubated with two primary antibodies targeting different histone modifications, followed by nanobody-Tn5 in situ transposition. The same tissue section was then labeled with a panel of barcoded poly-A-tailed oligo-conjugated antibodies, which recognize surface antigens. Next, in situ reverse transcript was performed using the Biotinylated poly-T RT primer to capture complimentary DNA of both poly-A-tailed oligo-conjugated antibodies and mRNA. The subsequent steps for spatial barcoding and library construction were similar to above discussed methods. The protein library and RNA library can be separated by SPRI bead purification. Frozen tissue slides were thawed for 1 min at 37 °C. Tissue was fixed with formaldehyde (0.2%, with 0.05 U μl–1 RNase Inhibitor) for 5 min and quenched with 1.25 M glycine for a further 5 min. After fixation, tissue was washed twice with 1 ml of 0.5× DPBS-RI and cleaned with ddH2O. The sequential order for spatial multiple profiling is as follows: 1. ATAC-seq; 2. Nanobody-based CUT&Tag; 3. In situ reverse transcription; 4. Ligation of barcode A; 5. Ligation of barcode B; 6. Reverse crosslink; 7. gDNA and cDNA separation; 8. Library construction; 9. Library QC and sequencing.