title: GSM2612306: NCH644 transduced with Control shRNA biological replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: Libraries for next-generation sequencing were prepared with total RNA with the TruSeq® RNA Sample Preparation Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer´s instructions. Poly-A RNA was purified from the total RNA preparation with magnetic oligo-dT beads. The RNA-bead mixture was incubated at 65°C for five minutes to denature the RNA. Then, the mixture was incubated for five minutes at RT to allow the RNA to bind to the beads. Afterwards, the beads were washed with bead washing buffer and resuspended in elution buffer for two minutes at 80°C. Bead binding buffer was added to allow rebinding of the eluted RNA. The beads were washed with bead washing buffer and resuspended in elute, prime, fragment mix. The RNA was eluted at 94°C for eight minutes, and first strand cDNA synthesis was performed with SuperScript II (Thermo Fisher Scientific, Waltham, MA, USA) in first strand mix supplied by Illumina. Second strand synthesis was performed with the second strand mix for one hour at 16°C and the resulting double stranded cDNA was purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). Then, end repair was performed for 30 minutes at 30°C with the provided end repair mix. The end repaired DNA was again cleaned up with AMPure XP beads. A-tailing was performed with the A-tailing mix at 37°C for 30 minutes followed by 70°C for five minutes. Indexed adapters were ligated to the end-repaired A-tailed cDNA at 30°C for ten minutes. Next, stop ligation buffer was added, and the libraries were cleaned up with AMPure XP beads. PCR amplification was performed with the provided PCR reagents and the following cycling conditions: Denaturation at 98°C for 30 seconds and then 15 cycles of1) 98°C for 10 seconds, 2)60°C for 30 seconds and 3)72°C for 30 seconds. Afterwards, a final extension at 72°C for five minutes was performed and the amplified libraries were purified again with AMPure XP beads.