GSM6025788: SAM24381185 Thyroid aJ1aJ2-3 (pool of 6 mice); Mus musculus; RNA-Seq
Thyroid tissue was extracted from mice at the time of necropsy. To generate single cells, thyroids were cut with a razon blade and digested for 30min at 42°C. The digestion was done with 0.05mg/ml Liberase TM (Sigma, 5401127001) diluted in Advanced DMEM/F12, 10mM HEPES, 1% Glutamax, 1% P/S and 1:1000 Rock inhibitor (Y27632, StemCell 72304). After digestion, centrifuge the cells at 1200rpm/5min, wash them with Advanced DMEM/F12, 10mM HEPES, 1% Glutamax, 1% P/S and filter them through a 70um strainer. After digestion and wash, cells were centrifuged at 1200rpm/5min and resuspended in PBS 3% FBS. Given the low number of cells obtained from a single mouse thyroid, the thyroids from six mice were pooled into one sample. Chromium Single Cell 3' Library and Gel bead kit v2, following the manufacturer's manual (CG00052 Chromium Single Cell 3 Reagent Kits v2 User Guide RevA; 10x Genomics). Cell density and viability of the single-cell suspensions were determined by Vi-CELL XR cell counter (Beckman Coulter). All of the processed samples had more than 85% viable cells. Cell density was used to impute the volume of single-cell suspension needed in the RT master mix, aiming to achieve ~8000 cells per sample. cDNAs and libraries were prepared following the manufacturer's manual (10X Genomics). Libraries were profiled by Bioanalyzer High Sensitivity DNA kit (Agilent Technologies) and quantified using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA). Each library was sequenced in one lane of HiSeq4000 (Illumina) following the manufacturer's sequencing specification (10X Genomics).