GSM8769117: Intervertebral disc, Wild-type, replicate 4; Mus musculus; RNA-Seq
Flash-frozen IVDs were pulverized and reconstitued in TRIzol. Samples were quadruple filtered via column extraction and centrifugation and remained in DNA/RNA-free water until analysis. Library preparation was performed with 1-50ng of total RNA.  ds-cDNA was prepared using the SeqPlex RNA Amplification Kit (Sigma) per manufacturer's protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. 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 index tags.