title: GSM8145202: MLNM15; Homo sapiens; OTHER
library_construction_protocol: The tumor areas were marked by referring to the H&E staining of the consecutive slide, with the accordance of a certificated pathologist. The estimated tissue areas were measured using Image J software. Then, the optimal amounts of tissue for each HTG EdgeSeq AI assay were scraped and placed into microfuge tubes. The calculated volume of lysis buffer for each collected sample was added, and the samples were then overlaid with 500 μL of HTG denaturation oil. After centrifugation, the samples were incubated at 95 °C for 15–20 min to denature protein structures and remove paraffin wax from the FFPE tissue sections. Samples were cooled down for 10 min at room temperature (RT), and HTG provided Proteinase K was pipetted into the aqueous (lysis, non-oil) phase of the samples at a volume 1/20th of the total lysis buffer. Finally, the samples were incubated at 50 °C for 3 h, with the aqueous phase being mixed by pipetting every 30 min. Subsequently, 25 μL of the sample lysate was loaded onto the HTG EdgeSeq instrument for probe-capture for 20 h. Probe-captured FFPE samples for HTG-AI assay were amplified and indexed via polymerase chain reaction (PCR) using the master mix (OneTaq HotStart 2X MasterMix in GC Buffer (New England Biolabs Inc., Ipswich, MA, USA), and indexing primers. The PCR reaction conditions were as follows: (1) 95 °C for 4 min, (2) 95 °C for 15 s, (3) 56 °C for 45 s, (4) 68 °C for 45 s, (5) repeating steps 2–4 for a total of 19 cycles, (6) 68 °C for 10 min, and (7) holding at 4 °C. Following PCR, library cleanup was performed with a mixture of clean up buffer (39% of 5M NaCl, 31.25% of 40% PEG 8000, 29.75% of molecular-grade water) and AMPure XP beads, by combining it with the PCR-amplified sample at 5:2 ratio, respectively. After 5 min of incubation, the samples were placed on the magnetic stand, then washed twice with 80% ethanol, followed by a 5-min air drying period. The elution was performed using 40 μL of 10mM Tris-HCl, pH 8.0. All libraries were quantitated using the KAPA Library Quant Kit (Illumina Inc., San Diego, CA, USA) and the Universal qPCR Mix Kit (Roche, Basel, Switzerland) in accordance with the manufacturer's recommendations. Quality checks (QC) for library size were performed on the Agilent Technologies TapeStation 2200 instrument using the High Sensitivity D1000 ScreenTape and High Sensitivity D1000 reagents (Agilent Technologies Inc., Santa Clara, CA, USA). The expected peak size ranged between 150 and 170 base pairs. Samples that did not indicate proper library formation were excluded from sequencing and the library preparation process was repeated. Quantitated libraries were diluted, normalized, and pooled based on the raw quantity (pM) generated by the HTG EdgeSeq RUO Library calculator software version 2.0.0. Then, libraries were denatured in 0.2 N NaOH for 8 min at RT. NaOH was hydrolyzed with 200mM Tris pH 7.4, and the denatured pool was then diluted down to 4 pM using the Hyb buffer supplied in the NextSeq 550 High Output Kit v2.5 (Illumina Inc., San Diego, CA, USA). To introduce sequencing diversity and a positive sequencing control, 4 pM of 12.5 pM PhiX Control v3 (Illumina Inc., San Diego, CA, USA) was spiked into the diluted and denatured 4 pM sample pool. The final pool consisted of 95% sample libraries and 5% PhiX control by volume. The pooled library was then denatured at 98 °C for 4 min and immediately placed on ice for at least 5 min before loaded onto the Illumina NextSeq 550 instrument, following the Illumina instrument sequencing protocol. Sequencing on Illumina platforms was conducted according to the HTG instructions, with a read length of 1 × 50 base pairs. The raw sequencing data was transformed into FASTQ files using Illumina BaseSpace BCL to FASTQ software version 2.2.0 and Illumina Local Run Manager Software version 2.0.0. FASTQ files were analyzed with HTG EdgeSeq Parser software version v5.1.724.4793 to generate raw counts for a total of 2,002 genes. Each sample was profiled for 2,002 genes related to tumor-immune interaction, in addition to 18 control genes.