DSP-1001660009393-B-C06
Library strategy was using the NanoString GeoMx Digital Spatial Profiler (DSP) transcriptional assays. DSP collection sample plates were dried down by sealing the plates with aeroseal and placed at RT overnight. The samples were then resuspended with diethyl pyrocarbonate (DEPC)-treated water. 96-well plates were prepared by mixing 2 L polymerase chain reaction (PCR) mix (NanoString), 4 L of index primer mix (NanoString), and 4 L of DSP sample. The following PCR program was used to amplify the Illumina sequencing compatible libraries: 1) 37C for 30 minutes, 50C for 10 minutes, and 95C for 3 minutes; 2) 18 cycles of 95C for 15 seconds, 65C for 1 minutes, and 68C for 30 seconds; 3) 68C for 5 minutes and a final hold at 4C. The indexed libraries were pooled into 8-well strip tubes, and incubated with 58 L AMPure XP beads (Beckman Coulter, Cat# A63880) in a 1:1.2 ratio. The 8-well strip was placed on a magnetic stand before removal of the supernatant. The beads were then washed twice with 80% ethanol and dried before being eluted with 10 L elution buffer (10mM Tris-HCl pH 8, 0.05% Tween-20). The samples were combined into two pools of 40 L each and incubated with 48 L AMPure buffer (2.5 M NaCl, 20% PEG 8000) for 5 minutes. Following magnetic incubation for 5 minutes and removal of supernatants, the beads were washed twice with 80% ethanol and dried for 1 minute before elution with 17 L elution buffer. The two pools were combined to a final elute of 34 L. Sequencing library size was assessed with a DNA high sensitivity bioanalyzer assay (Agilent Technologies). Library concentration was assessed with Qubit dsDNA high sensitivity assay (Thermo Fisher Scientific). Total target counts per DSP collection plate for sequencing were calculated from the total sampled areas (m2) reported in the DSP generated Lab Worksheet. All sequencing libraries were generated with unique indexes allowing pooled sequencing. WTA libraries for sequencing with an Illumina NovaSeq 6000 platform.
SUB14931522
DSP-1001660009393-B-C06