GSM5503576: LGr RT353; Homo sapiens; RNA-Seq
HGSC and SBT tissues were obtained from the FRQS tissue bank (Université de Sherbrooke), the Ontario Tumor Bank (OTB) and the Alberta Research Tumor Bank (ARTB). Each 30 mg tissue sample was homogenized in 1 mL of TRIzol Reagent (Ambion) using a Polytron tissue homogenizer. RNA was isopropanol precipitated and resuspended in water and stored at −80°C. RNA integrity and concentration was assessed using the 2100 Bioanalyzer (Agilent). The prepared libraries were ribodepleted using the Illumina Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat). The ribodepleted RNA was purified with Zymo RNA Clean & Concentrator using a modified form of the protocol to conserve small RNAs (8X sample volume of 95-100% ethanol instead of 3X). Nonfragmented, ribodepleted RNA was then used as input for TGIRT template-switching reactions with 1 µM TGIRT-III RT (InGex, LLC) for 15 min at 60°C, as previously described (Qin et al. 2016). During the template-switching reaction, a starting molecule composed of a DNA oligonucleotide encoding the reverse complement of an Illumina Read 2 sequencing primer binding site annealed to an RNA oligonucleotide such that there is a single nucleotide 3' DNA overhang. This overhang (an equimolar mix of A, T, G, C) anneals to the 3' end of a target RNA molecule, which upon addition of dNTPs, causes TGIRT to initiate cDNA synthesis, resulting in seamless addition of the adapter sequence to the 5' end of the cDNA. After cDNA cleanup with QIAGEN MinElute Enzyme Reaction Cleanup kit, purified cDNAs are then ligated at their 3' ends to a 5'-adenylated DNA oligonucleotide encoding the reverse complement of an Illumina Read 1 sequencing primer binding site. Finally, index and capture sequences required for Illumina sequencing are added by PCR (12 cycles). Libraries were size-selected with AMPure XP beads (Beckman Coulter) and library quality was evaluated using an Agilent 2100 Bioanalyzer. TGIRT-seq libraries were sequenced on an Illumina HiSeq 4000 or 2500 instrument (PE 2x150 and PE 2x125, respectively) at the Genome Sequencing and Analysis Facility (GSAF) at the University of Texas at Austin. TGIRT-Seq