GSM5196005: Ctrl_0hr; Homo sapiens; RNA-Seq
Samples were prepared for scRNAseq using the 10X Genomics Next GEM Single cell 3' Reagent Kit v3.1. Single cell suspensions were washed in 1X PBS (calcium and magnesium free) containing 0.04% weight/volume BSA (400 μg/mL) and brought to a concentration of ~700-1200 cells/μL. This was accomplished by staining 10 μL of the single cell suspension with Trypan blue and reading on the Countess™ II Automated Cell Counter (ThermoFisher). The appropriate volume of cells was loaded with Single Cell 3' Gel Beads into a Next GEM Chip G and run on the Chromium™ Controller. GEM emulsions are incubated and then broken. Silane magnetic beads were used to clean up GEM reaction mixture. Read one primer sequence was added during incubation and full-length, barcoded cDNA is then amplified by PCR after cleanup. Sample size was checked on a Tapestation 4200 (Agilent) using the DNAHS 5000 tape and concentration determined by the Qubit 4.0 Fluorimeter (ThermoFisher) using the DNA HS assay. Samples were enzymatically fragmented and underwent size selection before proceeding to library construction. During library preparation, Read 2 primer sequence, sample index and both Illumina adapter sequences were added. Subsequently, samples were cleaned up using Ampure XP beads (Beckman Coulter) and post library preparation quality control was performed using the DNA 1000 tape on the Agilent Tapestation 4200. Final concentration was ascertained using the Qubit DNA HS assay. Samples are loaded at 1.6 pM and run on the Illumina NextSeq500 High Output FlowCell using V2.5 chemistry. Run configuration is 28x98x8.