GSM3590958: slice 3, tumor; Homo sapiens; RNA-Seq
Total RNA was extracted using the Qiagen miRNeasy mini kit (Qiagen Inc.). Samples were processed by the Illumina FFPe TruSeq RNA ACCESS library prep kit. A single adenine was added to 3' blunt end fragments using an A-tailing Mix for ligation to adapters with a single thymine end. The 30 µl reaction comprised 1.5 µl of resuspension buffer for each sample (100 ng) plus 12.5µl of A-tailing mix followed by incubation (37°C, 1 h) and denaturation (70°C, 5 min). Adapters which serve as linkers to the flow cell surface were ligated to the ds cDNA fragments (P5: 5' end; P7:3' end). Unique bar code indices were added as discriminating sample identifiers. Resuspension buffer (2.5 µl), an RNA Adapter Index (2.5 µl) and Ligation Mix (2.5 µl) were added to each sample bringing the volume to 37.5 µl for incubation (30°C, 15 min). Stop Ligation Buffer (5 µl) was added followed by AmpXP beads (42 µl, 10 min, RT) and the cDNA eluted using the Dyna Mag 2 magnet (5 min, RT) as previously described. A 15 cycle PCR enrichment step was performed to select fragments with adapters on both ends by combining Primer Cocktail (5 µl) and PCR Master Mix (25 µl) with each sample (20 µl) followed by denaturation, selective annealing and extension in a thermocycler (30 sec @ 98°C; [15 X: 10 sec @ 98°C, 30sec @ 60°C, 30sec @ 72°C]; 5 min @ 72°C; 4°C Hold). The samples were transferred to 1.5 ml LoBind tubes, mixed with AMPure beads (50 µl XP beads, 10 min incubation) and separated using the Dyna-Mag 2 magnet (5 min). The supernatant from each sample was discarded and the cDNA subjected to two EtOH washes (200 µl of 80% EtOH, 30 sec, 2X) as described previously followed by elution in 17.5 µl Resuspension Buffer.