title: GSM5236429: Exophytic melanoma mROI 13 (HTA-MELATLAS-1); Homo sapiens; RNA-Seq
library_construction_protocol: 40 μm regions of interest were extracted using a mechanical picker from 5 μm tissue section. Tissue microregions were deposited with 2 µl PBS into PCR tubes containing 18 µl of lysis buffer: 1:16 mix of Proteinase K solution (QIAGEN) in PKD buffer (QIAGEN), with 0.1% RNasin Plus. After deposit, tubes were immediately placed in dry ice and stored at -80°C until ready for downstream RNA-seq workflow. PCR tubes containing tissue microregions in the lysis buffer were removed from the freezer, allowed to thaw at room temperature for 5 min, and incubated at 56°C for 1 hr. Tubes were briefly vortexed, spun down, and placed on ice. Dynabeads Oligo(dT)25 beads (ThermoFisher) were washed three times with ice-cold 1X hybridization buffer (NorthernMax buffer (ThermoFisher) with 0.05% Tween 20 and 0.0025% RNasin Plus), and resuspended in original bead volume with ice-cold 2x hybridization buffer (NorthernMax buffer with 0.1% Tween 20 and 0.005% RNasin Plus). A volume of 20 µl of washed beads was added to each lysed sample, mixed by pipette, and incubated at 56°C for 1 min followed by room temperature incubation for 10 min. Samples were placed on a magnet and washed twice with an ice-cold 1X hybridization buffer, then once with ice-cold 1X PBS with 0.0025% RNasin Plus. Supernatant was removed, and the pellet was resuspended in 10.5 µl nuclease-free water. Samples were incubated at 80°C for 2 min and immediately placed on a magnet. Supernatant was transferred to new PCR tubes or plates, and placed on ice for subsequent whole transcriptome amplification or stored at -80°C. Reverse transcription and cDNA amplification were performed using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio, Kusatsu, Shiga, Japan). Resulting amplified cDNA libraries were assessed for DNA concentration using the Qubit dsDNA HS Assay Kit (ThermoFisher) and for fragment size distribution using the BioAnalyzer 2100 High Sensitivity DNA Kit (Agilent). Melanoma tissue microregion libraries were prepared with ThruPLEX DNA-seq Kit (Takara Bio). Resulting libraries were characterized by using the Qubit dsDNA HS Assay Kit and BioAnalyzer 2100 High Sensitivity DNA Kit, pooled at equimolar ratios, and sequenced using a NextSeq (Illumina) sequencer.