GSM9698048: dissected zebrafish intestines, homozygote mutants from pbx3a whole gene deletion heterozygous incross; Danio rerio; RNA-Seq
Dissected intestinal tissues were collected in 400 µL of sterile Ringer's solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES, pH 7.0) in 1.5 mL Eppendorf tubes. For each scRNA-seq experiment, protease solution (0.25% trypsin, 1 mM EDTA in 1× PBS, pH 8.0; Sigma-Aldrich T4549) was prepared fresh, and collagenase P (100 mg/mL in Hank's Balanced Salt Solution; Sigma-Aldrich H9269 and 11213865001) was thawed on ice. For each sample, 1.2 mL of protease solution was added to a well of a 24-well plate, which was then incubated at 28 °C to equilibrate. The Ringer's solution from each dissected sample (WT and mutant) was reduced to 100 µL, and the intestines were gently abraded by pipetting five times with a P200 set to 80 µL. Samples were rinsed by adding 1 mL fresh Ringer's solution, allowing tissue to settle, and removing all but 100 µL. The intestines, along with the remaining Ringer's, were then transferred into the equilibrated protease solution, followed by addition of 30 µL collagenase P per well. After swirling to mix (final volume 1.33 mL per well), plates were incubated at 28 °C and dissociation was monitored under a stereomicroscope. Every 5 minutes, samples were pipetted slowly 20 times with a P1000. After 10 minutes, each sample was passed once through a P200 by transferring from one well to another. Digestion was stopped after 20–25 minutes by adding 270 µL of 6× STOP solution (30% calf serum, 6 mM CaCl₂ in 1× PBS) and gently swirling (final volume 1.5 mL). Some small tissue fragments typically remained, but over-digestion usually reduced cell viability significantly and was avoided. Cell suspensions were then passed through a 40 µm cell strainer into 1.5 mL tubes, and each tube was flicked vigorously 20 times. Cells were spun down at 300 x g for 3 minutes at 4°C. Supernatant was removed (leaving ~ 100 L) and cells were resuspended in 1 mL chilled DMEM/F12 (0% BSA; Gibco 12500062). Cells were washed by spinning at 300 x g for 3 minutes at 4°C, removing supernatant, and resuspending cells in 1 mL chilled DMEM/F12 (0% BSA). The single cell suspension was then filtered once again through a 40 µm cell strainer into clean tube, centrifuged at 300 x g for 3 minutes at 4°C. Supernatant was removed leaving ~50 L in which the cell suspension was resuspended. Droplet emulsions of single cell capture were generated using the 10X Genomics Chromium controller with Single Cell 3' v3.1 consumable reagents for pbx3ay731 mutant samples, and the 10X Genomics Chromium GEM–X platform with Single Cell 3' v4 reagents for the meis1by730 mutant samples. Single-cell suspensions were stained with acridine orange / propidium iodide (10 mL of cells + 1 mL of Logos F23001 Acridine Orange/Propidium Iodide Stain) and then examined and quantified on a Logos Luna FL automated cell counter. Samples with sufficient concentration, low multiplet rate (<5% to proceed, <4.2% on average), and high viability (>85% to proceed, >90% on average) were then loaded into the instrument. WT and mutant samples for each mutant were then loaded into two channels at normal concentrations (pbx3ay731: 18,000 cells loaded, targeting 10,000 cells recovered; meis1by730: 15,000 –18,000 cells loaded, targeting 11,000 – 13,000 cells recovered). Downstream reactions were performed in Eppendorf MasterCycler x50s Thermal cyclers. 10–12 cycles were used for cDNA amplification, and the result was inspected using Agilent D5000 ScreenTape Tapestation Assay. The remaining steps were performed according to the 10X 3' v3.1 and 3' v4 protocols, with 8–12 cycles used for sample index PCR, based on the concentration of amplified cDNA in the previous step. Final libraries were evaluated using Agilent D5000 ScreenTape Tapestation Assay and quantitated using a ThermoFisher Qubit 4 with dsDNA High Sensitivity (HS) reagents. Average fragment length from the Bioanalyzer and concentrations obtained from the Qubit were used to pool the WT and mutant libraries in equimolar concentrations. Libraries were then sequenced on a Novaseq 6000 Sequencing System (Illumina) using SP100-cycle full flowcells with 28 bases for Read 1, 10 bases for i7 index, 10 bases for i5 index, and 90 bases for Read 2. PhiX control library was spiked in at 1%. Reads obtained from these runs were used for downstream analyses.