title: GSM7673547: atac_61_CC0216; Homo sapiens; OTHER
library_construction_protocol: Single-cell ATAC and RNA-seq libraries were prepared using the Chromium Single Cell Multiome ATAC + Gene Expression platform (10X Genomics, Pleasanton, CA). Briefly, cryopreserved PBMCs were thawed and processed for single nuclei multi-omics analysis according to the manufacturer's instructions (10x Genomics, CG000365 Rev B). Nuclei were obtained by incubating PBMCs for 3.10 minutes in freshly prepared Lysis buffer and washed and resuspended in chilled diluted nuclei buffer (10x Genomics, 2000153). About 9,000 cells were targeted for each experiment. Prepared nuclei were subjected to transposition of open chromatin regions. Next, transposed nuclei, reverse transcription Master Mix, barcoded Gel Beads, and Partitioning Oil were partitioned into single-cell GEMs (Gel Bead in EMulsions) using the 10X Chromium Controller and Next GEM Chip J. Within each GEM, Gel Beads are dissolved and poly-adenlyated (poly-A) mRNA transcripts are captured by uniquely barcoded poly(dt)VN oligos. Simultaneously, accessible chromatin fragments are captured by a separate oligo containing a spacer, unique barcode, and Illumina P5 adaptor sequence. The GEMs were then incubated in a C1000 Touch Thermal Cycler (BioRad) to produce barcodedDNA from the transposed DNA, and full-length cDNA. GEMs are then broken to release and pool single-cell fractions within each sample. Pooled fractions are purified using silane magnetic beads and subjected to PCR amplification to generate sufficient mass for library construction. Next, P7 and a sample index are added to transposed DNA via PCR to generate ATAC libraries. Finally, cDNA is enzymatically fragmented before the addition of P5, P7, i7 and i5 indexes, and TruSeq Read 2 via End Repair, A-tailing, Adaptor Ligation, and PCR resulting in gene expression libraries. Quantitation of gene expression and ATAC libraries was performed using Bioanalyzer High Sensitivity DNA Analysis (Agilent). Libraries were combined into gene expression and ATAC pools and sequenced on an Illumina NovaSeq 6000 system using the read lengths recommended by 10X: 28bp (read 1), 90bp (read 2), 10bp (i7 index), and 10bp (i5 index) for gene expression libraries and 50bp (read 1), 49bp (read 2), 8bp (i7 index) and 24bp (i5 index) for ATAC libraries. CellRanger v.3.1.0 (10xGenomics) was used to demultiplex raw sequencing data and quantify transcript levels against the 10x Genomics GRCh38 reference v.3.0.0.