title: GSM4105833: NF2 (Non-painful)_set1_N6; Homo sapiens; RNA-Seq
library_construction_protocol: Prior to the RNA extraction from Formalin-fixed, Paraffin-embedded (FFPE) tumor tissue slides (5-10µm sections), pathology revision was performed on a H&E stained tumor sections to confirm the tumor presence, histology, and grade of differentiation. Next, FFPE sections were de-paraffinized and re-hydrated in sequential steps and subjected to Cresyl violet (0.5% w/v) guided macro-dissection using RNAase free scalpel. This method was utilized to ensure at least 80% tumor cell content within the samples subjected to nucleic acid extraction. During RNA extraction, Cresyl violet stain was utilized as it minimizes the chemical modification of nucleic acids compared to H&E staining. FFPE RNA extraction was performed was using miRNeasy FFPE kit (Cat No. 217504, QIAGEN, USA) and DNase digestion was performed using RNase-Free DNase Set (Cat No. 79254 QIAGEN, USA) following manufacturer's instructions. Libraries were prepared using Illumina TruSeq stranded Total RNA sample preparation kit from 500ng of RNA. Prior to library prep, purified total RNA samples were evaluated for quality by Agilent Bioanalyzer to calculate RIN score and DV200. RNA samples with a RIN score > 7 as well as RNA samples with a RIN < 7 but DV200 > 50% were fragmented at 94C for 8min according to manufacturer's recommendation. RNA samples with RIN < 7 and DV200 < 50% were not fragmented. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Roche Kapa Biosystems library quantification kit according to manufacturer's protocols. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq500 with paired-end 75bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities.
attributes.tag: GEO Accession
attributes.value: GSM4105833