GSM6647333: 293747, 1_ATAC; Homo sapiens; RNA-Seq
Cryopreserved PBMCs were washed and counted using Countess II FL Automated Cell Counter (ThermoFisher). Nuclei were prepared from 600,000 cells by incubation and wash with chilled Lysis Buffer (10 mM Tris-HCl, pH 7.4 with 10mM NaCl, 3mM MgCl2, 0.01% Tween-20, 0.1% NP-40m 0.01% Digitonin, 1% BSA, 1mM DTT, and 1U/ul RNase inhibitor). Intact nuclei were collected by centrifugation, resuspended in 1X Diluted Nuclei Buffer (10X Genomics) with 1mM DTT and 1U/uL RNase inhibitor, and nuclei concentration and viability determined using Countess II FL with trypan blue. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.