title: GSM7006787: TC5-S11P-L: Wild-type; 26, 28, 30, 32, 34, 36 hpf; rep 1, low conc.; Danio rerio; RNA-Seq
library_construction_protocol: Per sample, 10–12 embryos or larvae were dechorionated using forceps in calcium-free Ringer's solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES, pH 7.0, 500 mL, sterile) with MESAB (400 ug/mL) and left to sit 5–10 minutes at 28°C to become anesthetized. Protease solution was mixed fresh each day (0.25% trypsin, 1 mM EDTA, pH 8.0, in 1xPBS, sterile; Sigma-Aldrich T4549). Per sample, 1.2 mL of protease solution was added to a well in a 24-well plate. This plate was placed at 28°C to equilibrate temperature. Meanwhile, collagenase P solution (100 mg/ml Collagenase P in Hank's Balanced Salt Solution; Sigma-Aldrich H9269 and 11213865001) was thawed on ice. Each sample of embryos/larvae were transferred to a 1.5mL Eppendorf tube, and the volume of Ringer's solution was reduced to 100 uL. The animals were de-yolked and abraded by pipetting up and down 5 times with a P200 set to 80 uL. Animals were rinsed by adding 1 mL of fresh Ringer's solution, allowing the animals to settle to the bottom, and removing all but 100 uL of the Ringer's solution. The animals (including the 100 uL of Ringer's) were then transferred into the 24-well plate with protease solution, 30 uL of Collagenase P solution was added per well, and the plate was swirled to mix (final volume = 1.33 mL per well). The plate was incubated at 28°C, monitoring dissociation with a microscope, pipetting up and down with a P1000 20 times slowly every 5 minutes. After 10 minutes, each sample was passed once through a P200 by pipetting from one well into another. Digestion was stopped after 20–25 minutes by adding 270 uL of 6X STOP solution (6X, 30% calf serum, 6 mM CaCl2, in 1 x PBS, sterile) to each well and swirling gently to mix (final volume per well now 1.5 mL). In many cases, some small chunks of tissue remained, but attempting to achieve complete digestion usually reduced cell viability significantly. Suspensions were filtered through a 40 uM cell filter into 1.5 mL Eppendorf tube to remove any remaining tissue chunks. The 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 ul) 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). During the spin, 11 uL of anchor solution was added to each well of a round-bottom plate (200 nM anchor oligo, 200 nM barcode oligo in DMEM/F12 with 0% BSA; anchor oligo was a gift from Chris McGinnis and the Zev Gartner lab, now available from Sigma-Aldrich; barcodes ordered from IDT). Cell suspension was distributed into 10-12 wells (100 uL cells per well), mixed with 5-10 gentle pipettings, then incubated on ice 5 minutes. To lock MULTI-seq barcodes in place, 11 uL of co-anchor oligonucleotide solution (200 nM co-anchor oligo in DMEM/F12 with 0% BSA; co-anchor oligo also a gift from Chris McGinnis and the Zev Gartner lab, now available from Sigma-Aldrich) was added to each well, pipetted gently to mix, and incubated on ice for 5 minutes. Labeling was halted with BSA by adding 50 uL of DMEM/F12 (4% BSA) and mixing gently. To remove excess labeling reagents, cells were washed twice by spinning cells down at 300 x g for 3 minute at 4°C, removing supernatant, and resuspending in 200 uL of DMEM/F12 (1% BSA). After the final wash, samples were pooled in a 2 mL Eppendorf, washed by spinning at 250 x g for 4 minutes, removing the supernatant, and resuspending in 1 mL of DMEM/F12 with 1% BSA, spinning again at 250 x g for 4 minutes, and then resuspending in 300 uL of DMEM/F12 with 1% BSA. Cells were filtered through a 40 uM FlowMi cell filter (Bel-Art H13680-0040) into a clean tube. Droplet emulsions of single cells were generated using the 10X Genomics Chromium controller with Single Cell 3' v3.1 consumable reagents, according to the manufacturer's instructions. In brief, single cell suspensions were stained with acridine orange / propidium iodide (11 uL of cells + 1 uL 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, <3.6% on average), and high viability (>85% to proceed, >95% on average) were then diluted with Ringer's to load into the instrument. Cells had been barcoded with MULTI-seq hash oligos to enable overloading of the instrument, which would then return of a larger number of transcriptomes with an increased rate of doublets that could then be removed computationally based on identification of multiple hash barcodes associated with that cell barcode. Samples were loaded into two channels at normal (9,600 cells loaded, targeting 6,000 cells recovered) and high (34,000 cells loaded, targeting 21,250 cells recovered) concentrations. Downstream reactions were performed in the Biorad C1000 Touch Thermal cyclers. [10–12 cycles] were used for cDNA amplification, and the result was inspected using Agilent High Sensitivity DNA Kits on the Agilent Bioanalyzer 2100. [8–13 cycles] were then used for sample index PCR, based on the concentration of amplified cDNA in the previous step. Final libraries were evaluated using Agilent High Sensitivity DNA Kits on the Agilent Bioanalyzer 2100 and quantitated using a ThermoFisher Qubit 4 with dsDNA High Sensitivity (HS) reagents. Average fragment length from the Bioanalyzer and concentration from the Qubit were used to pool libraries in equimolar concentrations. Libraries were sequenced across several sequencing runs. Some samples (including samples TC1-24, TC1-48, TC1-72, TC1-96, TC2-36, TC2-60, TC2-84, and TC3-48) were checked in three separate runs on a Nextseq 500 System (Illumina) with High Output 75 cycle kits, with 28 bases for Read 1, 8 bases for Index 1, and 56 bases for Read 2. Most sequencing was performed in three separate runs on a NovaSeq 6000 Sequencing System (Illumina), using S4 full flowcells, with 28 bases for Read 1, 8 bases for Index 1, and 91 bases for Read 2. PhiX control library was spiked in at 1%. Libraries were briefly analyzed and re-pooled after the second sequencing run to try to achieve similar reads/cell across the entire data. Reads from all runs above were used in downstream analysis. Most sequencing runs contained mixtures of libraries to measure gene expression and MULTI-seq barcodes. 10X Genomics Single-cell 3' v3.1 + MULTI-seq