GSM5575063: scRNA-seq_sample2; Mus musculus; RNA-Seq
For single-cell sequencing of the tissue, mouse thyroid was dissociated with collagenase I (Sigma-Aldrich) as described above and subsequently resuspended in TrypLE Express (GIBCO) pre-heated to 37°C and dissociated under repeated pipetting. For single-cell sequencing of TFCOs, organoid droplets were incubated in Cell Recovery solution (354253, Corning) for 30 minutes in order to dissolve the BME. Then, organoids were pelleted and resuspended in TrypLE Express (GIBCO) pre-heated to 37°C and dissociated under repeated pipetting. When the gland and the organoids were fully dissociated into single cells, samples were pelleted, washed, resuspended in FACS buffer (advanced DMEM/F12, 10 μM Y-27632 and DAPI) and strained (35 μm). DAPI-negative cells were immediately sorted into 384-well plates containing ERCC spike-ins (Agilent), RT primers and dNTPs (Promega) using a FACSFusion (BD Biosciences). Plates were prepared using Mosquito HTS (TTPlabtech). Single-cell RNA sequencing libraries were prepared following the SORT-seq protocol (Muraro et al., 2016) which is based on the CEL-seq2 method (Grün et al., 2015). In short, cells were first lysed for 5 minutes at 65 °C, and RT and second-strand mixes were dispensed by the Nanodrop II liquid handling platform (GC Biotech). Single-cell double-stranded cDNAs were pooled together and in vitro transcribed for linear amplification. Illumina sequencing libraries were prepared using the TruSeq small RNA primers (Illumina) and these DNA libraries were sequenced paired-end, respectively, on the Illumina NextSeq