snRNA-Seq of chicken: trunk embryos
24 trunks of embryos one day after electroporation with plasmid DNA: pCIG-MEK1ca 1 g/l + pCAGGS 1g/l (MEK1ca from human MEK1) were pooled, frozen in liquid nitrogen, and stored at  80C in subgroups of six samples each. Single nuclei were isolated using an in-house protocol.  Samples were then suspended in 2% BSA in PBS, filtered through 100 m cell strainers (VWR), centrifuged twice for 10 min at 500g, and resuspended in 2% BSA in PBS. Nuclei were incubated with an Alexa Fluor 647 anti-Nuclear Pore Complex Proteins Antibody Mab414 (BioLegend), then sorted using a FACSAria III (BD Biosciences) with the 85 m nozzle and the BD FACSDIVA software. Sorted nuclei were immediately processed on a Chromium Controller (10x Genomics). The mRNA was reverse-transcribed, converted to barcoded cDNA with sample indexing, purified using DynaBeads, and amplified by PCR. To construct the 5 gene expression library, the amplified barcoded cDNA was fragmented, end-repaired, poly-A-tailed, sample-indexed, and double-size selected with SPRI beads (average size 450 bp). The DNA was quantified, and fragment size distribution of the libraries was determined using the Qubit dsDNA HS assay kit (ThermoFisher, Q32851) and Agilent 2100 BioAnalyzer High Sensitivity DNA kit (Agilent Technologies, 5067-4626). Pooled libraries were then sequenced on an Illumina Nextseq 500 sequencing platform to a minimum sequencing depth of 20,000 reads per nucleus using read lengths of 26 bp read 1, 10 bp dual indexes, and 90 bp read 2.