title: GSM7646046: LAS250A947; Mus musculus; RNA-Seq
library_construction_protocol: Tissue was incubated with Collagenase I (1 mg/ml, Sigma, C0130) for 30-45 min and enzyme activity was terminated by rinsing cells with ice cold PBS. The cell suspension was centrifuged and filtered through 40 μm nylon cell strainers (Falcon, 352340). tdT+ cells were isolated by flow cytometry (BD Biosciences Aria Fusion sorter equipped with Diva V8 software) with a 100 μm nozzle and collected in 2% FBS in OptiMEM without phenol red (Thermo Fisher Scientific, 11058021). Dead cells were excluded by labelling with the cell viability dye Zombie Aqua (2 ng/ml, BioLegend, 423101) that was added to the single cell suspension before sorting. For the P0 stage, we also sorted the cells directly into 96-well plates to increase the yield. The SMART-Seq v4 Ultra Low Input RNA Kit for the Fluidigm C1 System, IFCs was used to reverse transcribe and amplify cDNA. cDNA was quantified using the Glomax (Promega). Libraries were prepared from cDNA generated from single cells using the Nextera XT kit from Illumina. Final libraries were pooled and sequenced on the HiSeq 4000.