RNASeq of homo sapien: adult female blood total RNA
Small RNA libraries were prepared using the Small RNA Library Prep Kit for Illumina (Norgen Biotek Corp., Canada). 6 uL of RNA from each sample were mixed to the 3' adapter and incubated at 70 C for 2 minutes. T4 RNA ligase 2 (truncated), buffer and RNase inhibitor were added to the mix and incubated for 1 hour at 28 C. Excess 3' adapters were removed using the provided column and the RNA was eluted in 12 uL. The 5' adapter was denatured for 2 minutes at 70 C and then mixed with 10 mM ATP, T4 RNA ligase 1 and the previously obtained elution, and was then incubated for 1 hour at 28 C. Reverse transcription was performed on the adapter ligation products by adding 10 mM dNTPs, first strand buffer and TruSCript reverse transcriptase and then incubated at 50 C for 1 hour. Indexing PCR was then performed by adding the PCR primer mix, PCR reverse primer and unique index for each sample. Final reaction volume was brought to 100 uL using nuclease-free water (Wisent Bioproducts, Canada) before amplifying in a 20-cycle PCR reaction.