title: GSM4411834: Six2 Positive; Mus musculus; RNA-Seq
library_construction_protocol: The female was sacrificed at day E18.5 of pregnancy and kidneys from eleven embryos were dissected, placed in PBS on ice, and examined under a fluorescent microscope to check for the presence of GFP. Kidneys from transgenic embryos showed a clear fluorescent pattern marking the cap mesenchyme, whereas those from the non-transgenic embryos showed uniform background fluorescence. Seven out of the eleven embryos contained the Six2-GFP transgene. Keeping the transgenic and non-transgenic kidneys separate, each kidney was then cut into 2-4 small pieces using a surgical razor blade and placed in 1ml of trypsin 0.25% (03-046-1A, Trypsin Solution B (0.25%), Biological Industries) using forceps. Initial tissue trituration was performed using a tissue grinder (D8938, Dounce, Large clearance pestle) followed by up-down pipetting with a P1000 pipette. Tissue fragments were then incubated with trypsin at 37C for 20 minutes, followed by additional trituration by pipetting. After visual confirmation that the majority of cells were fully dissociated, enzyme digestion was stopped by adding 2ml of DMEM with 10% FBS and placed on ice. Cells were first filtered with 70 micron cell strainers (CSS-010-070, Lumitron) and then 40 micron cell strainers (732-2757, VWR), pelleted by centrifugation for 5 minutes at 500 x g, re-suspended in 3ml PBS, and kept on ice until FACS sorting.    Single cell sorting was done using a BD FACSAria III flow cytometer with an 85 micron nozzle. Forward and side scatter were used to filter out red blood cells and to select for live single cells. Gating based on the negative control – the cells from the non-transgenic kidneys - was used to select for cells that were either positive (Six2-high) or negative (Six2-low) for expression of the Six2-GFP transgene. For single cell RNA sequencing, single cells were sorted into 96 individual wells from a 384 well plate that was pre-filled with Smartseq2 cell lysis buffer, RNase inhibitor, oligo-dT primer, ERCC, and dNTP's. Plates were spun for 1 minute to collect the liquid and cells at the bottom of the wells and immediately frozen. The Smartseq2 protocol was performed by the Israel National Center for Personalized Medicine (G-INCPM) as previously described with 22 amplification cycles. Altogether, 6 plates were processed, each containing 96 individual cells (576 cells total), with 3 plates containing Six2-high cells and 3 plates containing Six2-low cells. Each one of the 6 plates was separately sequenced 1 × 61 bases on the Illumina HiSeq 2500 platform in the Israel National Center for Personalized Medicine (G-INCPM). The Smartseq2 protocol
attributes.tag: GEO Accession
attributes.value: GSM4411834