GSM3109276: JV-01-Control-1: DMSO (0.01%), replicate 1; Homo sapiens; RNA-Seq
Total RNA was isolated from HT29 cells treated with vehicle and UroA (50 μM) (n=3) for 24 h and RNA was isolated using Trizol based lysis followed by Qiagen RNeasy kits.  The isolated RNA was checked for integrity (RIN>9.5) using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA) and quantified using a Qubit fluorometric assay (Thermo Fisher Scientific, Waltham, MA). Libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit- Set A (Cat# RS-122-2101) with poly-A enrichment. 1ug of samples (in a volume of 50 µl) were treated with RNA Purification Beads and denatured for 5 minutes at 65oC. Then the supernatant was discarded and the beads were washed with bead wash buffer. Captured polyadenylated RNA was eluted using Elution buffer at 80°C for 2 min. mRNA is further purified in a second bead clean-up, as well as fragmented and primed during elution by adding 19.5 µl of Elute, Prime, Fragment High Mix to the beads and incubating the samples for 8 minutes at 94oC. After fragmentation, 17ul of supernatant is removed from the beads and we proceeded immediately to synthesize first strand cDNA. Following the protocol, 8 µl of First Strand Synthesis Mix Act D and SuperScript II mix was added to each sample and heated on a thermocycler using preprogrammed thermal conditions. Once the reaction finished and reached 4oC, we immediately proceeded for second strand cDNA synthesis.  Diluted end repair control and Second Strand Marking Mix were added, mixed well and incubated in a pre-heated thermocycler at 16oC for one hour. Then the DNA was purified using Agencourt AMPure XP Beads. Finally samples were eluted with Resuspension Buffer and 15 µl of elute was collected and stored at -20oC. A-Tailing Control and A-Tailing Mix were added to the purified samples from step c and the samples were incubated on the preprogrammed thermal cycler. Once the incubation is done, we proceeded immediately to ligate adapters.  Diluted ligation control, Ligation Mix and barcodes were added and incubated in a pre-heated thermocycler at 30oC for 10 minutes. Stop Ligation Buffer was immediately added to each sample and mixed well.  Then the ligated samples were purified using Agencourt AMPure XP Beads. We eluted with 50 µl of Resuspension Buffer and the elute was again purified for a second time using Agencourt AMPure XP Beads. Afterwards, the final elution, consisting of 20 µl of the elute was collected and used for DNA enrichment. Samples were barcoded with Illumina TruSeq Adapters.
GEO Accession
GSM3109276