GSM8182662: cpc339_mir181_day7; Homo sapiens; RNA-Seq
At Day 7 of chondrogenic induction, 8 to 10 CPC Pellets were pooled, rinsed with DPBS, and submerged in a lysis buffer from Norgen BioTek's Total RNA Purification kit for 5 minutes. Pellets were then flash frozen in liquid nitrogen and dissociated using a handheld motorized pellet pestle. The flash freeze and pellet pestle process was repeated 3 to 5 times, or until the pellets were effectively homogenized. RNA was extracted from the dissociated pellets using Norgen BioTek's Total RNA Purification kit according to the manufacturer's instructions. Isolated total RNA was quantified using a NanoDrop 2000 Spectrophotometer. Total RNA integrity was determined using Agilent Bioanalyzer. Library preparation was performed with 500 ng to 1 µg of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94°C for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags.