title: GSM3204779: MS_WT1; Mus musculus; RNA-Seq
library_construction_protocol: Single cell suspensions from Plzf +/+ and Plzf lu/lu testes carrying the Eomes-tdTomato knockin allele were prepared using a two-step collagenase and trypsin enzymatic digestion procedure. Cells were filtered first with an 40um nylon membrane followed by a 25um filter. DNase I was added to the cell suspension to prevent clumping. Cells were washed once in PBS and finally suspended at a concentration of 1-2x106 cells/ml, analyzed and sorted on Becton Dickinson FACSVantage cell sorter. Cells from littermate mice that were negative for Eomes-tdTomato were used to gate the positive staining cell population and DAPI was used to gate out the dead cells. Single cells were flow sorted into individual wells of a Bio-Rad hard shell 384 well plate. The plate was immediately transferred and stored in a -80C freezer. Custom designed Drop-Seq barcodes from Integrated DNA Technologies (IDT) were delivered into each well of each 384 well plate. All primers in one well shared the same unique cell barcode and billions of different unique molecular identifiers (UMIs). An Echo 525 liquid handler was used to dispense 1ul of primers and reaction reagents into each well in the plate for the cell lysis and cDNA synthesis. Following cDNA synthesis, the contents of each well were collected and pooled into one tube using a Caliper SciClone Liquid Handler. After treatment with exonuclease I to remove unextended primers, the cDNA was PCR amplified for 13 cycles. The 10 cDNA was fragmented and amplified for sequencing with the Nextera XT DNA sample prep kit (Illumina) using custom primers (see below) that enabled the specific amplification of only the 3′ ends.