title: GSM6455112: BPS-MPE_100_175; Homo sapiens; RNA-Seq
library_name: GSM6455112
library_construction_protocol: Cell lysates were used directly for TempO-Seq library building TempO-Seq gene expression analysis was conducted on exposed and control MCF-7 cell lysates. Libraries were prepared in a 96-well plate format using the TempO-Seq® Human Whole Transcriptome Panel reagent kit (BioSpyder Technologies, Carlsbad, CA, USA), following the manufacturer?s protocol. Human Brain Total RNA and qPCR Human Reference Total RNA (Takara Bio, CA, USA) were included as positive assay controls and a no-lysate control (1X TempO-Seq® R Lysis Buffer only) was included as a negative assay control (two replicates per control). In brief, 2 µl cell lysates were hybridized with the targeted detector oligo (DO) probe mix in 1X TempO-Seq® R Lysis Buffer for 10 min at 70oC followed by a temperature gradient with a ramp rate of 0.5oC/min to 45oC over a 50 min period, followed by a nuclease digestion at 37oC for 90 min to remove excess, unbound, or incorrectly bound DOs enzymatically. Ligation of the DO pairs bound to adjacent target sequences was then completed with a 60 min incubation at 37oC, immediately followed with a 15 min enzyme denaturation at 80oC to generate a pool of amplification templates. All amplification templates (i.e., 10 µl of ligated DOs) were transferred to its corresponding well of the 96-well PCR Pre-Mix and Primers plate. Amplification proceeded with a CFX96 Real-Time PCR Detection System (Bio-Rad, Mississauga, ON, Canada) to attach a unique sequence tag and the sequencing adaptors to each sample using the following PCR program settings: 37oC for 10 min, 95oC for 2 min; 6 cycles of 95oC for 30 sec, 54oC for 30 sec, 72oC for 120 sec; 16 cycles of 95oC for 30 sec; 72oC for 2 min; 72oC for 1 min. The TempO-Seq® libraries were pooled (5 µl of all 96 samples) and purified using the Macherey-Nagel NucleoSpin® Gel and PCR Clean-Up kit (Clontech Laboratories Inc., Bethlehem, PA, USA), according to the manufacturer?s instructions for PCR cleanup with three modifications outlined in the TempO-Seq® Assay User Guide. The pooled, purified TempO-Seq® libraries were sequenced using a NextSeq® 500/550 High Output flow cell (v2 kit, 75 cycles) on an Illumina NextSeq® 500 Sequencing platform (Illumina, San Diego, CA, USA).