title: Discovery of traditional serrated adenoma by using DSP
design: Discovery of traditional serrated adenoma by using DSP
library_construction_protocol: All human tissue samples were obtained from the archive of Department of Pathology, National Taiwan University Hospital (NTUH). Hybridization cocktail with 25μl RNA probe mixture were made, containing 200μl buffer R (NanoString Technologies, Seattle, WA, USA) and 25μl DEPC water for each slide. We then applied the RNA probe mixture to each sample slide. Hybrislip (Thermo Fisher Scientific, Waltham, MA, USA) was used to cover to the sample slides. Then we incubated the slides in HyBEZ (ACDBio), (Advanced Cell Diagnostics, Inc., Newark, CA, USA) in a humid chamber at 37℃ for 16-24 h. On the next day, we washed the uncovered slides with prewarmed Stringent wash solution and SSC Solution (Sigma Aldrich, Inc. St. Louis, Missouri, USA). All the off-target probes were removed away from the slides. Then, we blocked the non-specific binding  with 200μl Buffer W (Nanostring Technologies, Seattle, WA, USA) for 30 minutes in the humidity DPEC-wetted chamber. After blocking, 220 µL of morphology marker mix was  applied to each sample and incubated for 1 hr. The tumours were visualized with staining using GeoMx Solid Tumor TME Morphology Kit (NanoString Technologies, Seattle, WA, USA) including 1:40x anti-CD45 (Yellow) plus PanCK (Green), and 1:10X SYTO 13 (nuclei, Blue) . Following the GeoMx DSP Instrument Quick Start Guide (MAN-10133-03), we prepared the PCR products with 2μl GeoMx Seq Code PCR Master Mix and 4 μl GeoMx DSP samples. CTA probe (Cat: GMX-RNA-NGS-CTA-4) was used as positive control and DEPC-water as negative control in this experiment. The following thermocycler programwas used: 37℃ incubation for 30 min, deactivation at 50℃ for 10 min, initial denaturation at 95℃ for 3 min cycles of denaturation, annealing and extension at 95℃, 65℃, and 68℃ for 15 sec, 60 sec, and 30 sec, respectively for  cycles, and final extension for the PCR product for 5 min. PCR products were then purified with two rounds of AMPure XP beads (Beckman Coulter, CA, USA) at 1.2X beads-to sample ratio. Libraries were paired-end sequenced on a NextSeq550. Up to 400 M total aligned reads were obtained. NextSeq550-derived FASTQ files for each sample were compiled for each compartment using the fastq program of Illumina, and then demultiplexed and converted to digital count conversion (DCC) by the GeoMx NGS Pipeline. The quality of the RNA library and NGS analysis were performed by the Medical Microbiota Center of the First Core Laboratory, National Taiwan University College of Medicine, Taipei, Taiwan. Additional files contain the H and E staining as well as the sample sequence codes.