title: GSM2171870: CTRL, rep1; Mus musculus; RNA-Seq
library_construction_protocol: E18.5 embryonic kidney pairs isolated from individual embryos were placed in PBS on ice, then transferred into 500 μl of 0.03% collagenase (Sigma, Cat#C1889) in PBS for 10 minutes at 37ºC. Samples were transferred to ice and the kidneys were dissociated by trituration using an 18 gauge needle with 8-10 passes followed by a 25 gauge needle for 8-10 passes to homogenize to a single-cell slurry. Cells were transferred to a 15ml falcon tube containing 4-5mls 2% fetal bovine serum (FBS) in PBS to inhibit collagenase. The cells were pelleted for 5 minutes at 400xg (4ºC) and washed three times in  5 mls of 2% fetal bovine serum (FBS). The final cell pellet was resuspended in 60ul 2% FBS in PBS and transferred to an Eppendorf tube. Cell count and viability was measured using 5 μl of cells + 5 μl trypan blue and a Hemocytometer. 5 μl of the remaining cell volume from each sample was pooled to serve as an unstained control. The remaining 50ul was processed for labelled with FITC-DBA lectin. The cells were pelleted at 400xg for 5 min. and stained in a fresh 50 μl of staining solution on ice and in the dark with DBA-FITC (Vector Labs, Cat#FL-1031) 1:10 diluted in 2% FBS in PBS for 20min. The cells were washed in 1mL of 2% FBS in PBS and centrifuged at 400 x g (4ºC) for 5min. The cell pellet was resuspended in 300-400 μl of 2% FBS in PBS for FACS. GFP positive gated cells were isolated using the unstained cells as a negative control. GFP+ cells were collected directly into 500 μl of Trizol (ThermoFisher) and snap frozen on dry ice and stored at -80ºC. RNA libraries were prepared for sequencing using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (mRNA) (New England Biolabs)
attributes.tag: GEO Accession
attributes.value: GSM2171870