Chromium single cell atac reagent kits

Nuclei isolation was performed following the protocol from 10× Genomics (CG000169). Briefly, 100 000–1 000 000 cells were collected by FACS and centrifuged at 300 g for 5 min at 4°C. Cell pellets were resuspended in 100 μl cold Lysis Buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 0.1% Nonidet P40, 0.01% Digitonin, 1% BSA), mixed by gently by pipetting then incubated for 2 min on ice. One milliliter of cold Wash Buffer (10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.1% Tween-20, 1% BSA) was then added immediately and mixed by gentle pipetting. The cells were then spun down at 500 g for 5 min at 4°C, and resuspended in cold 1× Nuclei Buffer (10× Genomics, PN-2000153). The nuclei concentration and quality were determined with a hemocytometer.
scATAC-seq library preparation was performed following the protocol from 10X Genomics (CG000168 Rev B) using the Chromium Single Cell ATAC Reagent Kits (10× Genomics). The libraries were cleaned using SPRIselect reagent (Beckman Coulter). The quantity and quality of the libraries were evaluated by the Qubit Fluorometer and Agilent Fragment Analyzer. The libraries were subsequently sequenced on a NovaSeq6000 (PE50).

Cryopreserved BMMCs from two donors were obtained from AllCells. One donor was a 93-year-old non-Hispanic white male with newly diagnosed chronic lymphocytic leukemia, and the other individual was healthy but otherwise unknown. Cells were isolated and lysed following the Nuclei Isolation for Single Cell ATAC Sequencing Demonstrated Protocol (10x Genomics: CG000169) following guidance for primary cells. Sequencing libraries were generated following the Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics: CG000168). Briefly, nuclei were tagmented in bulk. Single nuclei were encapsulated in GEMs containing a gel bead with a unique cell barcode, and genomic material was amplified in a linear amplification reaction. GEMs were broken, and genomic material was amplified via PCR appending a sample index and Illumina sequencing handles (P5/P7). Libraries were sequenced on the Illumina NovaSeq 6000 System according to the manufacturer’s protocols.

The cells from five cell lines were counted and mixed equally. Cell nuclei were isolated and washed according to the Nuclei Isolation for Single Cell ATAC Sequencing (10x Genomics) protocol, with 1 million cells to start with (0.2 million from each cell line) and 3 min lysis on 100μL buffer. Nuclei were then used to generate scATAC-seq libraries according to the Chromium Single Cell ATAC Reagent Kits User Guide (10x Genomics; CG000168 Rev B). Sequencing libraries were loaded on an Illumina sequencer with 2 × 75 paired-end kits using the following read length: 72 bp read 1, 8 bp i7 index, 16 bp i5 index, and 72 bp read 2. In the sequencing reaction, reads 1 and 2 contain the DNA insert, while the index reads, i5 and i7, capture the cell barcodes and sample indices, respectively. Cells were sequenced on Illumina HiSeq2500 with near around 300 million read pairs in total.