GSM7123490: Striatum, batch 1, single-cell multi-omics [scATAC-seq]; Mus musculus; ATAC-seq
For single nuclei preparation, frozen striatum were placed into 500 µl Miltenyi Nuclei Extraction Buffer (Miltenyi 130-128-024) plus 0.8 U/µl RiboLock RNase Inhibitor (ThermoFisher EO0382) in a gentleMACS C tube (Miltenyi 130-093-237) on ice, and then nuclei were immediately extracted in batches of 4 through the “4C_nuclei_1” program on the MACS Dissociator. Nuclei were placed on ice and filtered through a 70 µm pluriStrainer Mini into a pre-chilled 2 ml tube, and the C tube was washed with an additional 500 µl cold Miltenyi Nuclei Extraction Buffer and filtered. Nuclei were spun down at 350 rcf at 4 °C for 5 minutes and resuspended in 80 µl cold PBS with 2% BSA and 1 U/µl RiboLock. Nuclei were counted (~4,000 nuclei/µl) and 60,000 nuclei from each of the 8 samples were mixed in a chilled 1.5 ml Eppendorf tube. The pooled nuclei were spun at 300 rcf at 4 °C for 3 minutes and resuspended in 30 µl 10x Genomics Nuclei Buffer with 1 U/µl RNase inhibitor. The pooled nuclei were counted once more to confirm counts (~10,000 nuclei/ul), single-cell suspension, and lack of debris. Nuclei viability was assessed on a LUNA-FX7 automated cell counter (Logos Biosystems), and up to 40,000 nuclei (~5,000 from each sample) were loaded onto each of 4 lanes of a 10X Chromium microfluidic chip. Single nuclei capture and library preparation were performed using the 10X Chromium platform and according to the manufacturer's protocol (#CG000388 Chromium Next GEM Single Cell Multiome ATAC + Gene Expression). Because the 10X chip was superloaded, to reduce duplicate reads arising from multiple PCR steps, the number of cycles was adjusted from the 10X protocol. After GEM generation, barcoded cDNA and transposed DNA fragments were pre-amplified using 6 cycles. The pre-amplified sample was divided and used for two separate steps. An additional 6 cycles were used for adding a sample index for ATAC library construction and 12 PCR cycles for the gene expression library construction. cDNA and ATAC libraries were checked for quality on Agilent 4200 Tapestation and ThermoFisher Qubit Fluorometer, and quantified by KAPA qPCR, before sequencing; each gene expression library was sequenced using NovaSeq 6000 S4 v1.5 200 cycle flow cell lane, dual index scRNAseq asymmetric read configuration 28-10-10-90, targeting 20,000 nuclei with an average sequencing depth of 50,000 read pairs per nucleus. Each ATAC library was sequenced using Illumina NovaSeq 6000 S2 v1.5 100 cycle flow cell lane, with a 50-8-24-49 read configuration, also targeting 20,000 nuclei with an average sequencing depth of 50,000 reads per cell.