title: GSM8687026: IC3635,  REP3; Homo sapiens; RNA-Seq
library_name: GSM8687026
library_construction_protocol: Tumors were collected when tumor volume was 0.3~0.6 cm3.Tumor bearing mice were euthanized with CO2, tumor tissue was collected from three mice and put in 2ml cryogenic vial and flash frozen in liquid nitrogen. Tumor tissue was ground to a fine powder under liquid nitrogen in a cooled mortar and pestle. Tissue powder was transferred to dry ice-cooled cryogenic tube. Twenty to ~50mg tissue powder was measured and transferred to a precooled 1.5ml eppendorf tube, and RNA isolated by adding 1ml Trizol solution (ThermoFisher), and mixing immediately.  300l chloroform was added to the lysate mixture, and vortexed vigorously for 1min, and then left at room temperature for 5 minutes. The following procedure was performed as instructed in the manual of RNeasy Kit (QIAGEN). The quality of total RNAs was checked by Agilent Fragment Analyzer (Agilent Technologies, Santa Clara, CA), and only samples with RQN >7 were used for subsequent mRNA-seq library preparation and sequencing. Approximately 500ng total RNA was used for stranded mRNA-seq library preparation by following the NEB Directional mRNA-seq sample preparation guide (New England Biolabs, Ipswich, MA). The first step in the workflow involved purifying poly-A containing mRNA using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. These cDNA fragments then went through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products were then purified and enriched by PCR amplification to create the final RNA-seq library. RNA-seq libraries were subjected to quantification process and subsequent 100bp paired read sequencing run with Illumina NovaSeq 6000 platform at 100 bp paired-end module (Greehey Children's Cancer Research Institute's Genome Sequencing Facility at UTHSCSA).