GSM9314767: Duodenum; Homo sapiens; RNA-Seq
Fresh peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque density-gradient centrifugation and cryopreserved. At a later time, PBMCs were thawed. Gastrointestinal biopsies were dissociated into single cell suspensions using gentleMACS Tumor Dissociation Kit (Miltenyi Biotec). For both PBMC and GI samples, dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec). Viable cells were resuspended in 0.04% BSA in DPBS (no Ca2+/Mg2+). Library construction was performed according to the manufacturer's instructions (Single Cell Next GEM 5' v2 protocol, 10x Genomics). Briefly, PBMCs were resuspended in the master mix and loaded 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 contained an Illumina R1 primer sequence, 10x barcode, and unique molecular identifier (UMI). The pooled barcoded DNA was then cleaned up using Silane Dynabeads, amplified by PCR, and then the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, Illumina R2 primer sequences, paired-end constructs with P5 and P7 sequences, and a sample index were added.