GSM6586533: NSACB12_70.8_+S9_2; Homo sapiens; OTHER
Cell lysates were used directly for TempO-Seq library building The TempO-Seq® Human Surrogate+Tox Panel (S1500+) v2.0 (BioSpyder Technologies, Carlsbad, CA, USA) assay was completed following the manufacturer's instructions in a 96-well plate format. Each 96-well plate included three assay controls in duplicate: a negative no-cell lysate (1X TempO-Seq® Enhanced Lysis buffer only), Human Reference Total RNA (Takara Bio, CA, USA), and Human Brain Reference Total RNA (Takara Bio, CA, USA). Briefly, for each treatment, 2 µL of cell lysate in 1X TempO-Seq® Enhanced Lysis buffer was hybridized to the Human S1500+ Surrogate detector oligo (DO) probe mix (V2.0), incubated at 70°C for 10 minutes, then ramped down to 45°C over 50 minutes (0.5°C/minute). A nuclease digestion followed to remove excess or incorrectly bound DOs at 37°C for 1 hour. The bound DO pairs were then ligated together at 37°C for 1 hour and 15 minutes at 80°C to generate templates for amplification. A 10 µL aliquot of amplification template was transferred to its respective well in the TempO-Seq® PCR Pre-Mix and Primers plate and amplified on the CFX96 thermocycler (Bio-Rad, Mississauga, ON, Canada) with the following program: 37°C for 10 min, 95°C for 1 min; 25 cycles of 95°C for 10 sec, 65°C for 30 sec, 68°C for 30 sec; 68°C for 2 min. For library building and purification, three 96-well plates (288 sample libraries) were pooled together (5 µL per sample) and purified using the Macherey-Nagel NucleoSpin® Gel and PCR Cleanup Kit (Clontech Laboratories Inc., Bethlehem, PA, USA) with the adjustments specified by the TempO-Seq® Assay User Guide. The pooled purified TempO-Seq® libraries were then diluted, quantified, and assessed for quality using the Agilent High Sensitivity D1000 TapeStation (Agilent Technologies, Santa Clara, CA, USA) and the qPCR KAPA Library Quantification Kit (Universal qPCR Master Mix) for Illumina NextSeq 500. Samples were sequenced on a total of two NextSeq® 500/550 High Output (75-cycle) flow cells using an Illumina NextSeq® 500 Sequencing Platform (Illumina, San Diego, CA, USA). A separate pool was completed for a fourth plate and an additional 96 sample libraries were added to the sequencing data for subsequent analysis. The TempO-Seq® Human Surrogate+Tox Panel (S1500+) v2.0 - Index Sets F, G, H were used to index libraries (in two pools)