GSM5945979: YAP_2; Mus musculus; RNA-Seq
Thyroid tumors were dissected and placed in ice-cold digestion media (HBSS, 5%FBS, 10mM HEPES, 1.5mg/ml Collagenase A, 4mg/mL DNase I) and minced. The desegregated tissue was collected in digestion media and incubated at 37C for 1hour, briefly vortexing every 15 minutes. The cells were passed through 70 µM filter to remove tissue debris and obtain single cell suspension and washed with PBS. The cells were resuspended in FACS buffer (HBSS, 5%FBS, 10mM HEPES) and sorted by +Kate using a BD FACS Aria flow cytometer and ~20,000 Kate positive cells were collected into TRIzol LS (Invitrogen, #10296010) for RNA-sequencing. The total RNA was amplified using SMART-Seq v4 Ultra Low Input RNA Kit (Clonetech catalog # 63488), with 12 cycles of amplification. The amplified cDNA was used to prepare libraries with the KAPA Hyper Prep Kit (Kapa Biosystems KK8504) using 8 cycles of PCR, barcoded, and run on a HiSeq 4000 in a PE50 run using the HiSeq 3000/4000 SBS Kit (Illumina).