GSM8404796: Lefty2KO_75; Mus musculus; RNA-Seq
Embryos were isolated from surrogate mice. Outermost extraembryonic tissues (yolk sac, trophectoderm derived tissues) were preserved if possible. Undamaged embryos isolated experiment were washed in 1xPBS/0.4%BSA, pooled without pre-selection and subjected to tissue dissociation in 200 µl TrypLE Express (Gibco) for 20-30 minutes at 37ºC, with pipetting in 5 minute intervals. The cell suspension was filtered using Scienceware Flowmi Cell Strainers, 40 µM. Cells were washed twice with 1ml 1xPBS/0.4%BSA and centrifugation for 5 minutes at 1200 rpm. The cell concentration was determined using a hemocytometer and cells were subjected to single-cell RNA sequencing (10x Genomic, Chromium™ NextGem 3' v3.1) with dual barcodes. Single-cell libraries were generated following the manual instructions of 10x Genomic (Chromium™ Single Cell 3' v3), with the exception of fewer PCR cycles in cDNA amplification and sample index PCR than recommended in order to increase complexity of libraries. Libraries were sequenced with a minimum of 230 Mio paired end reads and parameters as described in the manual.