GSM7290737: E21.5 Nitrofen Control; Rattus norvegicus; RNA-Seq
Pooled NC and CDH specimens combined fetuses from two separate pregnant dams, and the two sets of pooled 2HC specimens each came from a different mother. Pooled lungs were digested into a single cell suspension, as previously described 46. Briefly, lung tissue was minced and dissociated in 0.1% collagenase A (Sigma Aldrich), 0.04% DNase I (Sigma Aldrich), and 0.5 mM CaCl2 in 3 ml of 10 mg/mL Dispase II (Sigma Aldrich. Incubations were performed on an orbital shaker at 37°C for a total of 45 min. Cell clumps were disrupted via pipetting, and the suspension was passed through a 40-micron cell strainer to remove any undigested tissue pieces or aggregates. Pelleted cells were then resuspended in 1% BSA in PBS to prevent   aggregation. Cell viability and concentration were assessed using propidium iodide and acridine orange with a CellDrop automated cell counter (DeNovix, Wilmington, DE). A quantity of 10,000 single cells were targeted using the 10x Genomics Chromium Controller (Pleasanton, CA) for cDNA synthesis and barcoding. cDNA quality and quantity of each sample were assessed using a Bioanalyzer High Sensitivity DNA assay, and samples were constructed following the 10x Genomics Chromium Single Cell 3' V3 protocol (CG000183 Rev A). Sample libraries were then assessed for proper construction using the same Bioanalyzer assay. Samples were pooled equally, and the resulting DNA library was quantified using the sparQ Universal Library Quantification kit (Quantabio, Beverly, Massachusetts) to inform sample preparation for downstream Illumina sequencing. Libraries were denatured and sequenced on   an Illumina Novaseq 6000 (San Diego, CA) high-throughput sequencing platform with 28 cycles for the forward read and 91 cycles for the reverse read. Each cell was sequenced to a minimum of 20,000 reads per cell per manufacturer's recommendations. We achieved an average sequencing saturation of 33.1%.