title: GSM4432589: Sample_2_Six2neg_14_6_TRI; 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. Six2-high and Six2-low cells were sorted into separate 1.5 ml tubes, each containing RNA purification buffer. This was repeated in three experiments in which we used two types of RNA purification kits: the “single cell RNA Purification Kit” (51800, Norgen Biotek) was used in one experiment where we sorted 50,000 Six2-high and 50,000 Six2-low cells, and the Direct-zol RNA MiniPrep (R2050, Zymo Research) was used in two experiments where we sorted approximately 100,000 Six2-high cells and 400,000 Six2-low cells. Bulk total RNA was purified according to the manufacturer's instructions and stored in -80C. Altogether, 6 samples of total RNA were collected (3 replicates, each containing total RNA from Six2-high and Six2-low cells).    Sequencing libraries were prepared using the INCPM-Genomics in-house protocol for mRNA sequencing. Briefly, the poly-A fraction (mRNA) was purified from 80-280 ng of total RNA, followed by fragmentation and generation of double-stranded cDNA. Then, end repair, A-tailing, adapter ligation, and PCR amplification steps were performed. Libraries were evaluated using a Qubit (Thermo fisher scientific) and TapeStation (Agilent). Sequencing libraries were constructed with barcodes to allow for multiplexing of 6 libraries in a single lane. All 6 libraries were sequenced paired-end 2 × 126 bases on an Illumina HiSeq 2500 platform in the Israel National Center for Personalized Medicine (G-INCPM). For each sample we obtained 35-42 million reads. The INCPM-Genomics in-house protocol for mRNA sequencing, which is compatible with Illumina's TrueSeq RNA sequencing protocol (with a few improvements)
attributes.tag: GEO Accession
attributes.value: GSM4432589