GSM8830071: Tuberous Sclerosis Mice Renal Cystadenomas (biol rep 1); Mus musculus; RNA-Seq
Excised renal tissues/tumors were rinsed twice with ice-cold phosphate-buffered saline (PBS), minced with fine, sterile razors, and incubated in digestion buffer (2 mg/ml collagenase I + 10ug/ml DNase I + elastase (0.075 U/ml) + 10mM EDTA dissolved in DMEM), at 37°C in a shaker at 300 rpm for 45 mins and then again for 30mins after vigorously trituration. Samples were centrifuged at 200 x g for 3 minutes at room temperature, and pellets were resuspended in 1x red blood cell lysis buffer for 10 minutes at room temperature. Lysis buffer is neutralized by mixing an equal volume of cell sorting media (PBS + 0.5% FBS) to the suspension, followed by centrifugation for 3 min at 200 x g at 4°C. Tissue pellets were resuspended in cell sorting media and passed sequentially through 100µm, 70µm, and 40µm cell strainers. After counting, cells were incubated with sytox green viability dye (1:1000 (v/v)) for 10 minutes in the dark at 4°C prior to cell sorting by flow cytometry using a BD FACS Aria II cell sorter (Becton Dickinson). Sytox Green (-) fractions per sample with cell viability of >85% were adjusted to 1000 cells/µl concentration before sequencing. Cell suspensions were sequenced using the 10x Genomics 3' Single Cell Gene Expression (GEX) workflow. Gel bead partitioning and cellular barcoding were done in the 10x Genomics Chromium Single Cell Controller, and library construction was performed using Illumina Single Cell 3' v3 chemistry according to the Chromium Single Cell 3' GEM library & gel bead kit protocol (version 3; 10x Genomics). Single-cell transcriptome libraries were pooled and sequenced on a NovaSeq6000 System (Illumina).