GSM9689607: 616E OS, FI, Day 7; Oryctolagus cuniculus; RNA-Seq
Following rabbit euthanasia with intravenous injection of sodium pentobarbital (120 mg/kg body weight), excised 8mm diameter central corneal buttons were digested in a 37 °C incubator (5 % CO2) with 2D rocking for approximately 3 h in 1 mL of media containing 2 mg/mL collagenase, and 0.5 mg/mL hyaluronidase in minimum essential medium (MEM) supplemented with 2 % penicillin/streptomycin/amphotericin. Following incubation, the solution underwent centrifugation (1500 rpm, 4 min), the supernatant was discarded, and the pellet was resuspended in 1 mL 0.05 % trypsin for incubation at 37 °C for 3 min. The trypsinization reaction was halted with 4 mL of trypsin inhibitor. After centrifugation (1500 rpm, 4 min), the supernatant was discarded, and the pellet was washed in 1 mL 4 °C chilled PBS. The cell suspension was filtered sequentially with 70 μm and 40 μm porosity Flowmi® cell strainers. Following centrifugation (1500 rpm, 4 min), the supernatant was discarded, and the single cells were resuspended in 500 μL 4 °C chilled PBS enriched with 0.4 % bovine serum albumin. Ice-cold, freshly prepared single cell suspensions were submitted for sequencing. Single cell suspensions are 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 is 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 is 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 are used to clean up GEM reaction mixture. Read one primer sequence is added during incubation and full-length, barcoded cDNA is then amplified by PCR after cleanup. Sample size is checked on the Agilent Tapestation 4200 using the DNAHS 5000 tape and concentration determined by the Qubit® 4.0 Fluorimeter from ThermoFisher using the DNA HS assay. Samples are enzymatically fragmented and undergo size selection before proceeding to library construction.  During library preparation, Read 2 primer sequence, sample index and both Illumina adapter sequences are added.  Subsequently, samples are cleaned up using Ampure XP beads and post library preparation quality control is performed using the DNA 1000 tape on the Agilent Tapestation 4200. Final concentration is ascertained using the Qubit DNA HS assay. Samples are then loaded at 650 pM on the NextSeq 2000  using a P2-100 flowcell with the run configuration of 28x90x10x10.