GSM9156651: NKM-1, SF153 1.2; Homo sapiens; RNA-Seq
Total RNA was extracted using the Kapa RNA HyperPrep Kit with RiboErase (KR1351). Using the Kapa RNA HyperPrep Kit with RiboErase (KR1351), rRNA is depleted starting from 25-1000ng of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA.  Samples are then fragmented using heat and magnesium.  1st strand synthesis is performed using random priming.  2nd strand synthesis incorporates dUTPs into the 2nd strand cDNA.  Adapters are then ligated, and the library is amplified.  Strands marked with dUTPs are not amplified allowing for strand-specific sequencing.  Indexed libraries that meet appropriate cut-offs for both quantity and quality are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul are used for sequencing.