GSM8442032: hBMSC, non-transfected, rep1; Homo sapiens; RNA-Seq
Total RNA was extracted using the Total RNA Purification Kit (Norgen Biotek Corp). Samples were DNAse I treated (Invitrogen, Thermo Fisher), and used for libreary preparation. Total RNA integrity was determined using Agilent Bioanalyzer and library preparation was performed with 500 ng 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 degrees 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.